Methods associated with tumor burden for assessing response to a cell therapy

ABSTRACT

Provided herein are methods and articles of manufacture for use with cell therapy for the treatment of diseases or conditions, e.g., cancer, including for predicting and treating a toxicity. In some embodiments, the toxicity is related to cytokine release syndrome (CRS). The methods generally involve assessing a change in a factor indicative of tumor burden prior to administration of a cell therapy in a subject that is associated with and/or correlate to a risk of developing toxicity. In some aspects, the methods can be used to determine if the subject is at risk or likely at risk for developing a toxicity following administration of the cell therapy. Also provided are methods for treating a subject having a disease or condition, in some cases involving administration of the cell therapy, based on assessment of risk of developing a toxicity following administration of the therapy. Also provided herein are reagents and kits for performing the methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. provisional patentapplication 62/580,417, filed Nov. 1, 2017, entitled “METHODS ASSOCIATEDWITH TUMOR BURDEN FOR ASSESSING RESPONSE TO A CELL THERAPY,” thecontents of which are incorporated by reference in their entirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitled735042013040SeqList.txt, created Oct. 31, 2018, which is 35,571 bytes insize. The information in the electronic format of the Sequence Listingis incorporated by reference in its entirety.

FIELD

The present disclosure provides methods and articles of manufacture foruse with cell therapy for the treatment of diseases or conditions, e.g.,cancer, including for predicting and treating a toxicity. In someembodiments, the toxicity is related to cytokine release syndrome (CRS).The methods generally involve assessing a change in a factor indicativeof tumor burden in a subject, which is associated with and/or correlatesto a risk of developing toxicity following administration of the celltherapy. In some aspects, the methods can be used to determine if thesubject is at risk or likely at risk for developing a toxicity followingadministration of the cell therapy. The present disclosure also providesmethods for treating a subject having a disease or condition accordingto a particular treatment regimen, in some cases involvingadministration of the cell therapy, based on assessment of risk ofdeveloping a toxicity following administration of the therapy. Alsoprovided herein are reagents and kits for performing the methods.

BACKGROUND

Various methods are available for adoptive cell therapy using engineeredcells expressing recombinant receptors, such as chimeric antigenreceptor (CARs). Improved methods are needed, for example, to increasesafety and/or reduce the risk of toxicity in a subject to theadministered cells. Provided are methods, kits and articles ofmanufacture that meet such needs.

SUMMARY

Provided herein is a method of assessing a risk of a toxicity or atoxicity-related outcome, following administration of a cell therapy,the method including assessing a factor indicative of disease burden attwo time points prior to receiving a cell therapy from a subject that isa candidate for receiving a cell therapy for treatment of a disease orcondition; and determining a fold change in the factor indicative ofdisease burden between the two time points, wherein the fold changeindicates the risk or likely risk of the subject developing a toxicityfollowing administration of the therapy to the subject. In someembodiments, the factor indicative of disease burden is a volumetricmeasure of a tumor or is an inflammatory marker in a sample from asubject. In some cases, the factor indicative of disease burden is avolumetric measure and the volumetric measure is a sum of the productsof diameters (SPD), longest tumor diameters (LD), sum of longest tumordiameters (SLD), tumor volume, necrosis volume, necrosis-tumor ratio(NTR), peritumoral edema (PTE), and edema-tumor ratio (ETR). In someaspects, the volumetric measure is measured using computed tomography(CT), positron emission tomography (PET), and/or magnetic resonanceimaging (MRI) of the subject.

In some aspects, the factor indicative of disease burden is aninflammatory marker and the inflammatory marker is C-reactive protein(CRP), erythrocyte sedimentation rate (ESR), albumin, ferritin, β2microglobulin (β2-M), or lactate dehydrogenase (LDH). In someembodiments, the sample is or contains a blood sample, plasma sample, orserum sample. In some cases, the inflammatory marker is assessed using acolorimetric assay or an immunoassay. In some examples, the inflammatorymarker is assessed using an immunoassay and the immunoassay is selectedfrom enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay(EIA), radioimmunoassay (RIA), surface plasmon resonance (SPR), WesternBlot, Lateral flow assay, immunohistochemistry, protein array orimmuno-PCR (iPCR).

In some of any such embodiments, the subject is a human.

In some of any such embodiments, the two time points includes a firsttime point and a second time point, and wherein the fold change is aratio of the factor indicative of disease burden at the first time pointand the second time point. In some aspects, the two time points are bothno more than one month or two months prior to receiving the celltherapy. In some cases, the two time points are not less than one week,two weeks, three weeks, four weeks, or five weeks apart. In someembodiments, the two time points are not less than three weeks apart. Insome embodiments, the two time points are not more than four weeksapart, five weeks, or six weeks apart. In some instances, the secondtime point is more than 1, 2, 3, 4, 5, 6, or 7 days beforeadministration of the cell therapy.

In some of any such embodiments, the cell therapy contains cellsengineered to express a recombinant receptor. In some embodiments, thetoxicity is neurotoxicity and/or cytokine release syndrome (CRS). Insome aspects, the toxicity is early toxicity that develops within 7 daysof administration of the cell therapy. In some examples, the toxicitydevelops within 3, 4, 5, 6, or 7 days of administration of the celltherapy. In some instances, the toxicity is a first sign of a fever oris a sustained fever following administration of the cell therapy.

In some embodiments, the subject is or is likely at risk of developingtoxicity if the fold change is at or above a threshold value; or thesubject is not or is likely not at risk of developing toxicity if thefold change is below a threshold value. In some aspects, the thresholdvalue is a value that: i) is within 25%, within 20%, within 15%, within10%, or within 5% above the average fold change of the factor indicativeof disease burden and/or is within a standard deviation above theaverage fold change of the factor indicative of disease burden in aplurality of control subjects; ii) is above the highest fold change ofthe factor indicative of disease burden, optionally within 50%, within25%, within 20%, within 15%, within 10%, or within 5% above such highestfold change, measured in at least one subject from among a plurality ofcontrol subjects; and/or iii) is above the highest fold change asmeasured among more than 75%, 80%, 85%, 90%, or 95%, or 98% of subjectsfrom a plurality of control subjects.

In some embodiments, the plurality of control subjects are a group ofsubjects prior to receiving a cell therapy for treating a disease orcondition, said cell therapy containing cells genetically engineered toexpress a recombinant receptor, wherein each of the subjects of thegroup went on to develop toxicity, optionally early toxicity, optionallya fever or a sustained fever, within 7 days after receiving the celltherapy for treating the same disease or condition.

In some embodiments, if the subject is indicated as likely to developtoxicity, selecting the subject for administration of a therapeuticregimen, the therapeutic regimen comprising administering to thesubject: i. an agent or other treatment capable of treating, preventing,delaying, reducing or attenuating the development or risk of developmentof a toxicity and the cell therapy, wherein administration of the agentis to be administered (i) prior to, (ii) within one, two, or three daysof, (iii) concurrently with and/or (iv) at first fever following, theinitiation of administration of the cell therapy to the subject; ii. thecell therapy at a reduced dose or at a dose that is not associated withrisk of developing toxicity or severe toxicity, or is not associatedwith a risk of developing a toxicity or severe toxicity in a majority ofsubjects, and/or a majority of subjects having a disease or conditionthat the subject has or is suspected of having, following administrationof the cell therapy; and/or iii. the cell therapy in an in-patientsetting and/or with admission to the hospital for one or more days,optionally wherein the cell therapy is otherwise to be administered tosubjects on an outpatient basis or without admission to the hospital forone or more days; or iv. an alternative therapeutic treatment other thanthe cell therapy.

In some embodiments, if subject is indicated as likely not at risk ofdeveloping toxicity, selecting the subject for administration of atherapeutic regimen, the therapeutic regimen comprising administering tothe subject: i. the cell therapy, optionally at a non-reduced dose,optionally on an outpatient basis or without admission to the hospitalfor one or more days; ii. the cell therapy, wherein administration ofthe cell therapy does not include administering, prior to orconcurrently with administering the cell therapy and/or prior to thedevelopment of a sign or symptom of toxicity other than fever, an agentor treatment capable of treating, preventing, delaying, or attenuatingthe development of the toxicity; or iii. the cell therapy in anoutpatient setting and/or without admission of the subject to thehospital overnight or for one or more consecutive days and/or is withoutadmission of the subject to the hospital for one or more days.

In some embodiments, if the subject is indicated as likely to developtoxicity following administration of the cell therapy, the cell therapyis not administered to the subject.

In some embodiments, the method further includes administering thetherapeutic regimen to the selected subject.

Provided herein is a method of treatment including administering atherapeutic regimen to a subject that is a candidate for receiving acell therapy for treatment of a disease or condition, wherein theadministration is carried out following or based on the results ofassessing the subject for a fold change in a factor indicative ofdisease burden between two time points prior to receiving a celltherapy. In some aspects, the factor indicative of disease burden is avolumetric measure of a tumor or is an inflammatory marker in a samplefrom a subject. In some examples, the factor indicative of diseaseburden is a volumetric measure and the volumetric measure is a sum ofthe products of diameters (SPD), longest tumor diameters (LD), sum oflongest tumor diameters (SLD), necrosis, tumor volume, necrosis volume,necrosis-tumor ratio (NTR), peritumoral edema (PTE), and edema-tumorratio (ETR). In some cases, the volumetric measure is measured usingcomputed tomography (CT), positron emission tomography (PET), and/ormagnetic resonance imaging (MRI) of the subject.

In some embodiments, the factor indicative of disease burden is aninflammatory marker and the inflammatory marker is C-reactive protein(CRP), erythrocyte sedimentation rate (ESR), albumin, ferritin, β2microglobulin (β2-M), or lactate dehydrogenase (LDH). In some aspects,the sample is or contains a blood sample, plasma sample, or serumsample. In some cases, the inflammatory marker is assessed using acolorimetric assay or an immunoassay. In some examples, the inflammatorymarker is assessed using an immunoassay and the immunoassay is selectedfrom enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay(EIA), radioimmunoassay (RIA), surface plasmon resonance (SPR), WesternBlot, Lateral flow assay, immunohistochemistry, protein array orimmuno-PCR (iPCR).

In some of any such embodiments, the subject is a human.

In some of any such embodiments, the two time points includes a firsttime point and a second time point, and wherein the fold change is aratio of the factor indicative of disease burden at the first time pointand the second time point. In some embodiments, the two time points areboth no more than one month or two months prior to receiving the celltherapy. In some cases, the two time points are not less than one week,two weeks, three weeks, four weeks, or five weeks apart. In someinstances, the two time points are not less than three weeks apart. Insome cases, the two time points are not more than four weeks apart, fiveweeks, or six weeks apart. In some instances, the second time point ismore than 1, 2, 3, 4, 5, 6, or 7 days before administration of the celltherapy.

In some of any such embodiments, the cell therapy contains cellsengineered to express a recombinant receptor. In some embodiments, thefold change in the factor indicative of disease burden is associatedwith a risk of developing toxicity following administration of the celltherapy.

In some of any such embodiments, the toxicity is neurotoxicity and/orcytokine release syndrome (CRS). In some cases, the toxicity is earlytoxicity that develops within 7 days of administration of the celltherapy. In some aspects, the toxicity develops within 3, 4, 5, 6, or 7days of administration of the cell therapy. In some instances, thetoxicity is a first sign of a fever or is a sustained fever followingadministration of the cell therapy.

In some of any such embodiments, the assessing of the fold change in afactor indicative of disease burden between two time points includes acomparison to a threshold value, wherein the comparison indicates therisk or likely risk of the subject developing toxicity followingadministration of the cell therapy to the subject. In some of any suchembodiments, the fold change in the factor indicative of disease burdencorrelates to a risk that the subject is or is likely to developtoxicity following administration of the cell therapy when it isadministered. In some of any such embodiments, the subject is or islikely at risk of developing toxicity if the fold change is at or abovea threshold value; or the subject is not or is likely not at risk ofdeveloping toxicity if the fold change is below a threshold value. Insome examples, the threshold value is a value that: i) is within 25%,within 20%, within 15%, within 10%, or within 5% above the average foldchange of the factor indicative of disease burden and/or is within astandard deviation above the average fold change of the factorindicative of disease burden in a plurality of control subjects; ii) isabove the highest fold change of the factor indicative of diseaseburden, optionally within 50%, within 25%, within 20%, within 15%,within 10%, or within 5% above such highest fold change, measured in atleast one subject from among a plurality of control subjects; and/oriii) is above the highest fold change as measured among more than 75%,80%, 85%, 90%, or 95%, or 98% of subjects from a plurality of controlsubjects.

In some cases, the plurality of control subjects are a group of subjectsprior to receiving a cell therapy for treating a disease or condition,said cell therapy containing cells genetically engineered to express arecombinant receptor, wherein each of the subjects of the group went onto develop toxicity, optionally early toxicity, optionally a fever or asustained fever, within 7 days after receiving the cell therapy fortreating the same disease or condition.

In some of any such embodiments, if the assessing indicates the subjectis or is likely to develop toxicity following administration of the celltherapy, the therapeutic regimen includes administering to the subject:i. an agent or other treatment capable of treating, preventing,delaying, reducing or attenuating the development or risk of developmentof a toxicity and the cell therapy, wherein administration of the agentis to be administered (i) prior to, (ii) within one, two, or three daysof, (iii) concurrently with and/or (iv) at first fever following, theinitiation of administration of the cell therapy to the subject; ii. thecell therapy at a reduced dose or at a dose that is not associated withrisk of developing toxicity or severe toxicity, or is not associatedwith a risk of developing a toxicity or severe toxicity in a majority ofsubjects, and/or a majority of subjects having a disease or conditionthat the subject has or is suspected of having, following administrationof the cell therapy; and/or iii. the cell therapy in an in-patientsetting and/or with admission to the hospital for one or more days,optionally wherein the cell therapy is otherwise to be administered tosubjects on an outpatient basis or without admission to the hospital forone or more days; or iv. an alternative therapeutic treatment other thanthe cell therapy.

In some of any such embodiments, if the assessing indicates the subjectis not or is likely not to develop toxicity following administration ofthe cell therapy, the therapeutic regimen includes administering to thesubject: i. the cell therapy, optionally at a non-reduced dose,optionally on an outpatient basis or without admission to the hospitalfor one or more days; ii. the cell therapy, wherein administration ofthe cell therapy does not include administering, prior to orconcurrently with administering the cell therapy and/or prior to thedevelopment of a sign or symptom of toxicity other than fever, an agentor treatment capable of treating, preventing, delaying, or attenuatingthe development of the toxicity; or iii. the cell therapy in anoutpatient setting and/or without admission of the subject to thehospital overnight or for one or more consecutive days and/or is withoutadmission of the subject to the hospital for one or more days.

In some of any such embodiments, if the assessing indicates the subjectis or is likely to develop toxicity following administration of the celltherapy, the cell therapy is not administered to the subject.

In some of any such embodiments, the disease or condition is a cancer.In some examples, the cancer is a myeloma, lymphoma or leukemia. In somecases, the disease or condition is a B cell malignancy. In someinstances, the B cell malignancy is selected from acute lymphoblasticleukemia (ALL), chronic lymphoblastic leukemia (CLL), acute myelogenousleukemia (AML), chronic myelogenous leukemia (CML), non-Hodgkin lymphoma(NHL), or Diffuse Large B-Cell Lymphoma (DLBCL), or a subtype of any ofthe foregoing.

In some of any such embodiments, greater than or greater than about 30%,35%, 40%, or 50% of the subjects treated according to the method do notexhibit any grade of cytokine release syndrome (CRS) or neurotoxicity;and/or at least at or about 45, 50, 60, 65, 70, 75, 80, 85, 90, 95% orabout 100% of subjects treated according to the method do not exhibitsevere CRS, optionally grade 3 or higher, prolonged grade 3 or higher orgrade 4 or 5 CRS; and/or at least at or about 45, 50, 60, 65, 70, 75,80, 85, 90, 95% or about 100% of subjects treated according to themethod do not exhibit severe neurotoxicity, optionally grade 3 orhigher, prolonged grade 3 or higher or grade 4 or 5 neurotoxicity;and/or at least at or about 45, 50, 60, 65, 70, 75, 80, 85, 90, 95% orabout 100% of subjects treated according to the method do not exhibitcerebral edema.

In some of any such embodiments, prior to initiation of administrationof the dose of cells, the subject has not been administered an agent ortreatment capable of treating, preventing, delaying, reducing orattenuating the development or risk of development of a toxicity; and/orthe subject is not administered an agent or treatment for the treatmentor prevention or reduction or attenuation of a neurotoxicity and/or acytokine release syndrome or risk thereof, within a period of timefollowing administration of the dose, which period of time is optionallyat or about 1, 2, 3, 4, 5 days or is optionally at or about 6, 7, 8, 9,10, 11 days or is optionally 1 or 2 or 3 or 4 weeks; and/or the subjectis not administered an agent or treatment for the treatment orprevention or reduction or attenuation of a neurotoxicity and/or acytokine release syndrome or risk thereof, following administration ofthe dose, prior to or unless the subject exhibits a sign or symptom ofthe toxicity and/or prior to or unless the subject exhibits a sign orsymptom of the toxicity other than a fever, optionally wherein the feveris not a sustained fever or the fever is or has been reduced or reducedby more than 1° C. after treatment with an antipyretic; and/or theadministration and any follow-up is carried out on an outpatient basisand/or without admitting the subject to a hospital and/or without anovernight stay at a hospital and/or without requiring admission to or anovernight stay at a hospital, optionally unless or until the subjectexhibits a sustained fever or a fever that is or has not been reduced ornot reduced by more than 1° C. after treatment with an antipyretic.

In some of any such embodiments, prior to initiation of administrationof the dose of cells, the subject has not been administered an anti-IL-6or anti-IL-6R antibody, optionally tocilizumab or siltuximab, and/or hasnot been administered a steroid, optionally dexamethasone, the subjectis not administered an anti-IL-6 or anti-IL-6R antibody, optionallytocilizumab or siltuximab, and/or has not been administered a steroid,optionally dexamethasone, within a period of time followingadministration of the dose, which period of time is optionally at orabout 1, 2, 3, 4, 5 days or is optionally at or about 6, 7, 8, 9, 10, 11days or is optionally 1 or 2 or 3 or 4 weeks; and/or the subject is notadministered an anti-IL-6 or anti-IL-6R antibody, optionally tocilizumabor siltuximab, and/or has not been administered a steroid, optionallydexamethasone, following administration of the cell dose, prior to, orunless, the subject exhibits a sign or symptom of a toxicity, optionallya neurotoxicity or CRS, and/or prior to, or unless, the subject exhibitsa sign or symptom of a toxicity, optionally a neurotoxicity or CRS,other than a fever, optionally wherein the fever is not a sustainedfever or the fever is or has been reduced or reduced by more than 1° C.after treatment with an antipyretic; and/or the administration and anyfollow-up is carried out on an outpatient basis and/or without admittingthe subject to a hospital and/or without an overnight stay at a hospitaland/or without requiring admission to or an overnight stay at ahospital, optionally unless or until the subject exhibits a sustainedfever or a fever that is or has not been reduced or not reduced by morethan 1° C. after treatment with an antipyretic.

In some of any such embodiments, the administration is carried out on anoutpatient basis and/or without requiring admission to or an overnightstay at a hospital; and if the subject exhibits a sustained fever or afever that is or has not been reduced or not reduced by more than 1° C.after treatment with an antipyretic, the subject is admitted to thehospital or to an overnight stay at a hospital and/or is administered anagent or treatment for the treatment or prevention or reduction orattenuation of a neurotoxicity and/or a cytokine release syndrome orrisk thereof.

In some of any such embodiments, the cell therapy is or contains tumorinfiltrating lymphocytic (TIL) therapy. In some of any such embodiments,the cell therapy contains cells engineered to express a recombinantreceptor that specifically binds to an antigen associated with a diseaseor condition and/or expressed in cells associated with the disease orcondition. In some of any such embodiments, the cell therapy is anadoptive cell therapy. In some cases, the cells contain immune cells. Insome examples, the immune cells are or contains T cells or NK cells. Insome aspects, the immune cells are or contains T cells and the T cellscontain CD4+ and/or CD8+ T cells.

In some of any such embodiments, the cell therapy is a T cell therapycomprising genetically engineered cells expressing a recombinantreceptor. In some cases, the recombinant receptor is a T cell receptoror a functional non-T cell receptor. In some examples, the recombinantreceptor specifically binds to an antigen associated with a disease orcondition and/or expressed in cells associated with the disease orcondition. In some examples, the antigen is selected from among 5T4,8H9, avb6 integrin, B7-H6, B cell maturation antigen (BCMA), CA9, acancer-testes antigen, carbonic anhydrase 9 (CAIX), CCL-1, CD19, CD20,CD22, CEA, hepatitis B surface antigen, CD23, CD24, CD30, CD33, CD38,CD44, CD44v6, CD44v7/8, CD123, CD138, CD171, carcinoembryonic antigen(CEA), CE7, a cyclin, cyclin A2, c-Met, dual antigen, EGFR, epithelialglycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), EPHa2,ephrinB2, erb-B2, erb-B3, erb-B4, erbB dimers, EGFR VIII, estrogenreceptor, Fetal AchR, folate receptor alpha, folate binding protein(FBP), FCRL5, FCRH5, fetal acetylcholine receptor, G250/CAIX, GD2, GD3,gp100, Her2/neu (receptor tyrosine kinase erbB2), HMW-MAA, IL-22R-alpha,IL-13 receptor alpha 2 (IL-13Ra2), kinase insert domain receptor (kdr),kappa light chain, Lewis Y, L1-cell adhesion molecule (L1-CAM),Melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MART-1,mesothelin, murine CMV, mucin 1 (MUC1), MUC16, NCAM, NKG2D, NKG2Dligands, NY-ESO-1, O-acetylated GD2 (OGD2), oncofetal antigen,Preferentially expressed antigen of melanoma (PRAME), PSCA, progesteronereceptor, survivin, ROR1, TAG72, VEGF receptors, VEGF-R2, Wilms Tumor 1(WT-1), a pathogen-specific antigen.

In some embodiments, the antigen is or includes αvβ6 integrin (avb6integrin), B cell maturation antigen (BCMA), B7-H3, B7-H6, carbonicanhydrase 9 (CA9, also known as CAIX or G250), a cancer-testis antigen,cancer/testis antigen 1B (CTAG, also known as NY-ESO-1 and LAGE-2),carcinoembryonic antigen (CEA), a cyclin, cyclin A2, C-C Motif ChemokineLigand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44,CD44v6, CD44v7/8, CD123, CD133, CD138, CD171, chondroitin sulfateproteoglycan 4 (CSPG4), epidermal growth factor protein (EGFR), type IIIepidermal growth factor receptor mutation (EGFR vIII), epithelialglycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrinB2,ephrin receptor A2 (EPHa2), estrogen receptor, Fc receptor like 5(FCRL5; also known as Fc receptor homolog 5 or FCRH5), fetalacetylcholine receptor (fetal AchR), a folate binding protein (FBP),folate receptor alpha, ganglioside GD2, O-acetylated GD2 (OGD2),ganglioside GD3, glycoprotein 100 (gp100), glypican-3 (GPC3), G ProteinCoupled Receptor 5D (GPRC5D), Her2/neu (receptor tyrosine kinaseerb-B2), Her3 (erb-B3), Her4 (erb-B4), erbB dimers, Human high molecularweight-melanoma-associated antigen (HMW-MAA), hepatitis B surfaceantigen, Human leukocyte antigen A1 (HLA-A1), Human leukocyte antigen A2(HLA-A2), IL-22 receptor alpha(IL-22Rα), IL-13 receptor alpha 2(IL-13Ra2), kinase insert domain receptor (kdr), kappa light chain, L1cell adhesion molecule (L1-CAM), CE7 epitope of L1-CAM, Leucine RichRepeat Containing 8 Family Member A (LRRC8A), Lewis Y,Melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MAGE-A10,mesothelin (MSLN), c-Met, murine cytomegalovirus (CMV), mucin 1 (MUC1),MUC16, natural killer group 2 member D (NKG2D) ligands, melan A(MART-1), neural cell adhesion molecule (NCAM), oncofetal antigen,Preferentially expressed antigen of melanoma (PRAME), progesteronereceptor, a prostate specific antigen, prostate stem cell antigen(PSCA), prostate specific membrane antigen (PSMA), Receptor TyrosineKinase Like Orphan Receptor 1 (ROR1), survivin, Trophoblast glycoprotein(TPBG also known as 5T4), tumor-associated glycoprotein 72 (TAG72),Tyrosinase related protein 1 (TRP1, also known as TYRP1 or gp75),Tyrosinase related protein 2 (TRP2, also known as dopachrometautomerase, dopachrome delta-isomerase or DCT), vascular endothelialgrowth factor receptor (VEGFR), vascular endothelial growth factorreceptor 2 (VEGFR2), Wilms Tumor 1 (WT-1), a pathogen-specific orpathogen-expressed antigen, or an antigen associated with a universaltag, and/or biotinylated molecules, and/or molecules expressed by HIV,HCV, HBV or other pathogens. Antigens targeted by the receptors in someembodiments include antigens associated with a B cell malignancy, suchas any of a number of known B cell marker. In some embodiments, theantigen is or includes CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33,Igkappa, Iglambda, CD79a, CD79b or CD30.

In some embodiments, the antigen is or includes a pathogen-specific orpathogen-expressed antigen. In some embodiments, the antigen is a viralantigen (such as a viral antigen from HIV, HCV, HBV, etc.), bacterialantigens, and/or parasitic antigens.

In some embodiments, the CAR contains an extracellularantigen-recognition domain that specifically binds to the antigen and anintracellular signaling domain comprising an ITAM, wherein optionally,the intracellular signaling domain contains an intracellular domain of aCD3-zeta (CD3ζ) chain; and/or wherein the CAR further contains acostimulatory signaling region, which optionally contains a signalingdomain of CD28 or 4-1BB. In some aspects, the genetically engineeredcells contain T cells or NK cells. In some of any such embodiments, thegenetically engineered cells contain T cells, and the T cells containCD4+ and/or CD8+ T cells. In some cases, the T cells are primary T cellsobtained from a subject. In some of any such embodiments, the cells ofthe cell therapy are autologous to the subject. In some of any suchembodiments, the cells are allogeneic to the subject. In some of anysuch embodiments, the threshold fold change for SPD is about 5 fold, 6fold, 7 fold, 8 fold, or 9 fold. In some of any such embodiments, thethreshold value is a threshold value for fold change of a volumetricmeasure of disease burden that is SPD, wherein the fold change is atleast about or is about or is 5 fold, 6 fold, 7 fold, 8 fold, or 9 fold.

In some of any such embodiments, the cell therapy includes theadministration of from or from about 1×10⁵ to 1×10⁸ total recombinantreceptor-expressing cells, total T cells, or total peripheral bloodmononuclear cells (PBMCs), from or from about 5×10⁵ to 1×10⁷ totalrecombinant receptor-expressing cells, total T cells, or totalperipheral blood mononuclear cells (PBMCs) or from or from about 1×10⁶to 1×10⁷ total recombinant receptor-expressing cells, total T cells, ortotal peripheral blood mononuclear cells (PBMCs), each inclusive. Insome of any such embodiments, the cell therapy includes theadministration of no more than 1×10⁸ total recombinantreceptor-expressing cells, total T cells, or total peripheral bloodmononuclear cells (PBMCs), no more than 1×10⁷ total recombinantreceptor-expressing cells, total T cells, or total peripheral bloodmononuclear cells (PBMCs), no more than 0.5×10⁷ total recombinantreceptor-expressing cells, total T cells, or total peripheral bloodmononuclear cells (PBMCs), no more than 1×10⁶ total recombinantreceptor-expressing cells, total T cells, or total peripheral bloodmononuclear cells (PBMCs), no more than 0.5×10⁶ total recombinantreceptor-expressing cells, total T cells, or total peripheral bloodmononuclear cells (PBMCs).

Provided is a kit containing a cell therapy, said cell therapycomprising cells genetically engineered to express a recombinantreceptor; and instructions for administering the cell therapy to asubject, wherein the instructions specify: (1) assessing one or morefactors indicative of tumor burden in a subject that is a candidate fortreatment with the cell therapy, wherein the assessing is performed attwo time points prior to the subject receiving the cell therapy, and thefactor indicative of tumor burden is a volumetric measure of the tumoror is an inflammatory marker in a sample from a subject; and (2)administering the cell therapy to the subject based on the fold changein the factor indicative of disease burden between the two time points,wherein the fold change indicates the risk or likely risk of the subjectdeveloping toxicity following administration of the cell therapy to thesubject.

Also provided is a kit containing a cell therapy, said cell therapycomprising cells genetically engineered to express a recombinantreceptor; and instructions for administering the cell therapy to asubject having or suspected of having a tumor, wherein the instructionsspecify: (1) assessing, at two different time points, one or morefactors indicative of disease burden in a subject that is a candidatefor treatment with the cell therapy, wherein, at each of the two timepoints, the subject has not yet been administered the cell therapy andthe factors indicative of disease burden comprises a volumetric measureof the tumor or a level or amount of an inflammatory marker in a samplefrom the subject, wherein the fold change in the factor assessed at thetwo different time points indicates the degree of risk or likely risk ofthe subject developing toxicity following administration of the celltherapy to the subject; and (2) (i) if the fold change is at or below athreshold value, administering the cell therapy to the subject, or (ii)administering the cell therapy to the subject, or (iii) administeringthe cell therapy to the subject at an amount, dose, setting, time orfrequency that is based on the fold change. In some embodiments, the kitfurther comprises instructions for assessing the presence, level oramount of at least one of the inflammatory marker in two or more samplesobtained at two or more time points from a subject that is a candidatefor treatment with the cell therapy and determining a fold-change in thelevel or amount of the marker assessed at two of the two or more timepoints, wherein the fold change indicates the degree of risk or likelyrisk of the subject developing toxicity following administration of thecell therapy to the subject. In some embodiments, the kit furthercomprises instructions for (i) administering a therapeutic regimencomprising the cell therapy to the subject if the fold change is at orbelow a threshold value, (ii) administering the cell therapy to thesubject, or (iii) administering the therapeutic regimen to the subjectat an amount, dose, setting, time or frequency based on the fold changeof the at least one inflammatory marker.

Also provided is a kit containing a cell therapy, said cell therapycomprising cells genetically engineered to express a recombinantreceptor; and one or more reagents for assaying one or more factorindicative of disease burden, wherein the factor indicative of diseaseburden is an inflammatory marker selected from C-reactive protein (CRP),erythrocyte sedimentation rate (ESR), albumin, ferritin, β2microglobulin (β2-M), or lactate dehydrogenase (LDH). In someembodiments, the kit further includes instructions for assessing thepresence, level or amount of at least one of the inflammatory marker intwo or more samples obtained at two time points from a subject that is acandidate for treatment with the cell therapy and determining thefold-change in the factor between the two time points, wherein the foldchange indicates the risk or likely risk of the subject developingtoxicity following administration of the cell therapy to the subject. Insome cases, the kit further contains instructions for administering atherapeutic regimen comprising the cell therapy to the subject followingor based on the fold change of the at least one inflammatory marker.

Provided is a kit containing a cell therapy, said cell therapycomprising cells genetically engineered to express a recombinantreceptor; and instructions for administering a therapeutic regimencomprising the cell therapy following or based on the results ofassessing the subject for a fold change in a factor indicative ofdisease burden between two time points prior to receiving a celltherapy, wherein the subject is a candidate for treatment with the celltherapy. Also provided is a kit containing a cell therapy, said celltherapy comprising cells genetically engineered to express a recombinantreceptor; and (b) instructions for administering a therapeutic regimencomprising the cell therapy based on the results of assessing thesubject for a fold change in a factor indicative of disease burdenbetween two time points prior to receiving a cell therapy, wherein theinstructions specify (i) administering the cell therapy to the subjectif the fold change is at or below a threshold value, (ii) administeringthe cell therapy to the subject, or (iii) administering the therapeuticregimen to the subject at an amount, dose, setting, time or frequencybased on the fold change, and wherein the subject is a candidate fortreatment with the cell therapy. In some aspects, the factor indicativeof disease burden is a volumetric measure of a tumor or is aninflammatory marker in a sample from a subject. In some embodiments, thefactor indicative of disease burden is a volumetric measure and thevolumetric measure is a sum of the products of diameters (SPD), longesttumor diameters (LD), sum of longest tumor diameters (SLD), necrosis,tumor volume, necrosis volume, necrosis-tumor ratio (NTR), peritumoraledema (PTE), and edema-tumor ratio (ETR). In some cases, the volumetricmeasure is measured using computed tomography (CT), positron emissiontomography (PET), and/or magnetic resonance imaging (MRI) of thesubject.

In some embodiments, the kit contains reagents for detecting C-reactiveprotein (CRP), erythrocyte sedimentation rate (ESR), albumin, ferritin,β2 microglobulin (β2-M), or lactate dehydrogenase (LDH). In someaspects, the inflammatory marker is assessed using a colorimetric assayor an immunoassay. In some examples, the inflammatory marker is assessedusing an immunoassay and the immunoassay is selected from enzyme-linkedimmunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay(RIA), surface plasmon resonance (SPR), Western Blot, Lateral flowassay, immunohistochemistry, protein array or immuno-PCR (iPCR).

In some of any such embodiments, the sample is or contains a bloodsample, plasma sample, or serum sample. In some of any such embodiments,the subject is a human.

In some of any such embodiments, the two time points includes a firsttime point and a second time point, and wherein the fold change is aratio of the factor indicative of disease burden at the a first timepoint and a second time point. In some of any such embodiments, the twotime points comprises a first time point and a second time point, andwherein the fold change is a ratio of the factor indicative of diseaseburden at the first time point and the factor indicative of diseaseburden at the second time point. In some cases, if the cell therapy isto be administered to the subject, the two time points are both no morethan one month or two months prior to the subject receiving the celltherapy. In some cases, the two time points are both no more than onemonth or two months prior to receiving the cell therapy. In someaspects, the two time points are not less than one week, two weeks,three weeks, four weeks, or five weeks apart. In some examples, the twotime points are not less than three weeks apart. In some examples, thetwo time points are not more than four weeks apart, five weeks, or sixweeks apart. In some cases, the second time point is more than 1, 2, 3,4, 5, 6, or 7 days before administration of the cell therapy. In somecases, if the cell therapy is to be administered to the subject, thesecond time point is more than 1, 2, 3, 4, 5, 6, or 7 days beforeadministration of the cell therapy.

In some of any such embodiments, the instructions specify that the foldchange in the factor indicative of disease burden indicates the subjectis or is likely at risk of developing toxicity if the fold change is ator above a threshold value; or the instructions specify that the foldchange in the factor indicative of disease burden indicates the subjectis not or is likely not at risk of developing toxicity if the foldchange is below a threshold value.

In some embodiments, the threshold value is a value that i) is within25%, within 20%, within 15%, within 10%, or within 5% above the averagefold change of the factor indicative of disease burden and/or is withina standard deviation above the average fold change of the factorindicative of disease burden in a plurality of control subjects; ii) isabove the highest fold change of the factor indicative of diseaseburden, optionally within 50%, within 25%, within 20%, within 15%,within 10%, or within 5% above such highest fold change, measured in atleast one subject from among a plurality of control subjects; and/oriii) is above the highest fold change as measured among more than 75%,80%, 85%, 90%, or 95%, or 98% of subjects from a plurality of controlsubjects. In some cases, the plurality of control subjects are a groupof subjects prior to receiving a cell therapy for treating a disease orcondition, said cell therapy containing cells genetically engineered toexpress a recombinant receptor, wherein each of the subjects of thegroup went on to develop toxicity, optionally early toxicity, optionallya fever or a sustained fever, within 7 days after receiving the celltherapy for treating the same disease or condition. In some cases, theplurality of control subjects is a group of subjects having beenadministered a cell therapy for treating a disease or condition andhaving had the fold change of the factor indicative of disease burdenassessed, wherein each of the subjects of the group went on to developtoxicity, optionally early toxicity, optionally a fever or a sustainedfever, within 7 days after receiving the cell therapy for treating thesame disease or condition.

In some of any such embodiments, the instructions specify if the foldchange indicates the subject is or is likely to develop toxicity,selecting the subject for administration of a therapeutic regimen, thetherapeutic regimen comprising administering to the subject: i. an agentor other treatment capable of treating, preventing, delaying, reducingor attenuating the development or risk of development of a toxicity andthe cell therapy, wherein administration of the agent is to beadministered (i) prior to, (ii) within one, two, or three days of, (iii)concurrently with and/or (iv) at first fever following, the initiationof administration of the cell therapy to the subject, ii. the celltherapy at a reduced dose or at a dose that is not associated with riskof developing toxicity or severe toxicity, or is not associated with arisk of developing a toxicity or severe toxicity in a majority ofsubjects, and/or a majority of subjects having a disease or conditionthat the subject has or is suspected of having, following administrationof the cell therapy; and/or iii. the cell therapy in an in-patientsetting and/or with admission to the hospital for one or more days,optionally wherein the cell therapy is otherwise to be administered tosubjects on an outpatient basis or without admission to the hospital forone or more days; or iv. an alternative therapeutic treatment other thanthe cell therapy.

In some of any such embodiments, the instructions specify if the foldchange indicates the subject is not or is likely not at risk ofdeveloping toxicity, selecting the subject for administration of atherapeutic regimen, the therapeutic regimen comprising administering tothe subject: i. the cell therapy, optionally at a non-reduced dose,optionally on an outpatient basis or without admission to the hospitalfor one or more days; ii. the cell therapy, wherein administration ofthe cell therapy does not include administering, prior to orconcurrently with administering the cell therapy and/or prior to thedevelopment of a sign or symptom of toxicity other than fever, an agentor treatment capable of treating, preventing, delaying, or attenuatingthe development of the toxicity; or iii. the cell therapy in anoutpatient setting and/or without admission of the subject to thehospital overnight or for one or more consecutive days and/or is withoutadmission of the subject to the hospital for one or more days.

In some of any such embodiments, the instructions specify if the foldchange indicates the subject is or is likely to develop toxicity, thetherapeutic regimen is not administered to the subject.

In some cases, the instructions specify the administration is carriedout on an outpatient basis and/or without requiring admission to or anovernight stay at a hospital; and if the subject exhibits a sustainedfever or a fever that is or has not been reduced or not reduced by morethan 1° C. after treatment with an antipyretic, the subject is admittedto the hospital or to an overnight stay at a hospital and/or isadministered an agent or treatment for the treatment or prevention orreduction or attenuation of a neurotoxicity and/or a cytokine releasesyndrome or risk thereof.

In some of any such embodiments, the toxicity is neurotoxicity and/orcytokine release syndrome (CRS). In some cases, the toxicity is earlytoxicity, and in some aspects is early toxicity that develops within acertain number of days, such as within 7 days, of administration of thecell therapy. In some examples, the toxicity develops within 3, 4, 5, 6,or 7 days of administration of the cell therapy. In some instances, thetoxicity is a first sign of a fever or is a sustained fever followingadministration of the cell therapy.

In some of any such embodiments, the cell therapy is or includes tumorinfiltrating lymphocytic (TIL) therapy. In some of any such embodiments,the cell therapy contains cells engineered to express a recombinantreceptor that specifically binds to an antigen associated with a diseaseor condition and/or expressed in cells associated with the disease orcondition. In some cases, the cell therapy is an adoptive cell therapy.In some aspects, the cells contain immune cells. In some instances, theimmune cells are or contain T cells or NK cells. In some examples, theimmune cells are or contain T cells and the T cells contain CD4+ and/orCD8+ T cells. In some instances, the cell therapy is a T cell therapycomprising genetically engineered cells expressing a recombinantreceptor.

In some embodiments, the disease or condition is a cancer. In someinstances, the cancer is a myeloma, leukemia or lymphoma. In someaspects, the disease or condition is a B cell malignancy. In someexamples, the B cell malignancy is selected from acute lymphoblasticleukemia (ALL), chronic lymphoblastic leukemia (CLL), acute myelogenousleukemia (AML), chronic myelogenous leukemia (CML), non-Hodgkin lymphoma(NHL), or Diffuse Large B-Cell Lymphoma (DLBCL), or a subtype of any ofthe foregoing.

In some embodiments, the antigen is selected from among 5T4, 8H9, avb6integrin, B7-H6, B cell maturation antigen (BCMA), CA9, a cancer-testesantigen, carbonic anhydrase 9 (CAIX), CCL-1, CD19, CD20, CD22, CEA,hepatitis B surface antigen, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6,CD44v7/8, CD123, CD138, CD171, carcinoembryonic antigen (CEA), CE7, acyclin, cyclin A2, c-Met, dual antigen, EGFR, epithelial glycoprotein 2(EPG-2), epithelial glycoprotein 40 (EPG-40), EPHa2, ephrinB2, erb-B2,erb-B3, erb-B4, erbB dimers, EGFR vIII, estrogen receptor, Fetal AchR,folate receptor alpha, folate binding protein (FBP), FCRL5, FCRH5, fetalacetylcholine receptor, G250/CAIX, GD2, GD3, gp100, Her2/neu (receptortyrosine kinase erbB2), HMW-MAA, IL-22R-alpha, IL-13 receptor alpha 2(IL-13Ra2), kinase insert domain receptor (kdr), kappa light chain,Lewis Y, L1-cell adhesion molecule (L1-CAM), Melanoma-associated antigen(MAGE)-A1, MAGE-A3, MAGE-A6, MART-1, mesothelin, murine CMV, mucin 1(MUC1), MUC16, NCAM, NKG2D, NKG2D ligands, NY-ESO-1, O-acetylated GD2(OGD2), oncofetal antigen, Preferentially expressed antigen of melanoma(PRAME), PSCA, progesterone receptor, survivin, ROR1, TAG72, VEGFreceptors, VEGF-R2, Wilms Tumor 1 (WT-1), a pathogen-specific antigen.

In some embodiments, the antigen is selected from among αvβ6 integrin(avb6 integrin), B cell maturation antigen (BCMA), B7-H3, B7-H6,carbonic anhydrase 9 (CA9, also known as CAIX or G250), a cancer-testisantigen, cancer/testis antigen 1B (CTAG, also known as NY-ESO-1 andLAGE-2), carcinoembryonic antigen (CEA), a cyclin, cyclin A2, C-C MotifChemokine Ligand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33,CD38, CD44, CD44v6, CD44v7/8, CD123, CD133, CD138, CD171, chondroitinsulfate proteoglycan 4 (CSPG4), epidermal growth factor protein (EGFR),type III epidermal growth factor receptor mutation (EGFR vIII),epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40),ephrinB2, ephrin receptor A2 (EPHa2), estrogen receptor, Fc receptorlike 5 (FCRL5; also known as Fc receptor homolog 5 or FCRH5), fetalacetylcholine receptor (fetal AchR), a folate binding protein (FBP),folate receptor alpha, ganglioside GD2, O-acetylated GD2 (OGD2),ganglioside GD3, glycoprotein 100 (gp100), glypican-3 (GPC3), G ProteinCoupled Receptor 5D (GPRC5D), Her2/neu (receptor tyrosine kinaseerb-B2), Her3 (erb-B3), Her4 (erb-B4), erbB dimers, Human high molecularweight-melanoma-associated antigen (HMW-MAA), hepatitis B surfaceantigen, Human leukocyte antigen A1 (HLA-A1), Human leukocyte antigen A2(HLA-A2), IL-22 receptor alpha(IL-22Ra), IL-13 receptor alpha 2(IL-13Ra2), kinase insert domain receptor (kdr), kappa light chain, Licell adhesion molecule (L1-CAM), CE7 epitope of Li-CAM, Leucine RichRepeat Containing 8 Family Member A (LRRC8A), Lewis Y,Melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MAGE-A10,mesothelin (MSLN), c-Met, murine cytomegalovirus (CMV), mucin 1 (MUC1),MUC16, natural killer group 2 member D (NKG2D) ligands, melan A(MART-1), neural cell adhesion molecule (NCAM), oncofetal antigen,Preferentially expressed antigen of melanoma (PRAME), progesteronereceptor, a prostate specific antigen, prostate stem cell antigen(PSCA), prostate specific membrane antigen (PSMA), Receptor TyrosineKinase Like Orphan Receptor 1 (ROR1), survivin, Trophoblast glycoprotein(TPBG also known as 5T4), tumor-associated glycoprotein 72 (TAG72),Tyrosinase related protein 1 (TRP1, also known as TYRP1 or gp75),Tyrosinase related protein 2 (TRP2, also known as dopachrometautomerase, dopachrome delta-isomerase or DCT), vascular endothelialgrowth factor receptor (VEGFR), vascular endothelial growth factorreceptor 2 (VEGFR2), Wilms Tumor 1 (WT-1), a pathogen-specific orpathogen-expressed antigen, or an antigen associated with a universaltag, and/or biotinylated molecules, and/or molecules expressed by HIV,HCV, HBV or other pathogens. Antigens targeted by the receptors in someembodiments include antigens associated with a B cell malignancy, suchas any of a number of known B cell marker. In some embodiments, theantigen is or includes CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33,Igkappa, Iglambda, CD79a, CD79b or CD30.

In some embodiments, the antigen is or includes a pathogen-specific orpathogen-expressed antigen. In some embodiments, the antigen is a viralantigen (such as a viral antigen from HIV, HCV, HBV, etc.), bacterialantigens, and/or parasitic antigens.

In some of any such embodiments, the recombinant receptor is a T cellreceptor or a functional non-T cell receptor. In some embodiments, therecombinant receptor is a chimeric antigen receptor (CAR). In somecases, the CAR contains an extracellular antigen-recognition domain thatspecifically binds to the antigen and an intracellular signaling domaincomprising an ITAM, wherein optionally, the intracellular signalingdomain contains an intracellular domain of a CD3-zeta (CD3ζ) chain;and/or wherein the CAR further contains a costimulatory signalingregion, which optionally contains a signaling domain of CD28 or 4-1BB.

In some of any such embodiments, the kit further contains an agent orother treatment capable of treating, preventing, delaying, reducing orattenuating the development or risk of development of a toxicity. Insome embodiments, the agent or other treatment is or includes one ormore of a steroid; an antagonist or inhibitor of a cytokine receptor orcytokine selected from among IL-10, IL-10R, IL-6, IL-6 receptor, IFN7,IFNGR, IL-2, IL-2R/CD25, MCP-1, CCR2, CCR4, MIP1β, CCR5, TNFalpha,TNFR1, IL-1, and IL-1Ralpha/IL-1beta; or an agent capable of preventing,blocking or reducing microglial cell activity or function. In someexamples, the antagonist or inhibitor is or includes an agent selectedfrom among an antibody or antigen-binding fragment, a small molecule, aprotein or peptide and a nucleic acid. In some cases, the agent or othertreatment is an anti-IL-6 antibody or an anti-IL6 receptor antibody. Insome examples, the agent or other treatment is or includes an agentselected from among tocilizumab, siltuximab, clazakizumab, sarilumab,olokizumab (CDP6038), elsilimomab, ALD518/BMS-945429, sirukumab (CNTO136), CPSI-2634, ARGX-109, FE301 and FM101. In some instances, the agentor other treatment is or includes tocilizumab. In some cases, the agentor other treatment is or includes siltuximab. In some of any suchembodiments, the steroid is or includes dexamethasone.

In some of any such embodiments, the genetically engineered cellsinclude T cells, and the T cells contain CD4+ and/or CD8+ T cells. Insome cases, the T cells are primary T cells obtained from a subject. Insome embodiments, the cells of the cell therapy are autologous to thesubject. In some of any such embodiments, the cells are allogeneic tothe subject. In some embodiments, the threshold value for SPD is about 5fold, 6 fold, 7 fold, 8 fold, or 9 fold. In some embodiments, thethreshold value is a threshold value for fold change of a volumetricmeasure of disease burden that is SPD, wherein the fold change is atleast about or is about or is 5 fold, 6 fold, 7 fold, 8 fold, or 9 fold.

DETAILED DESCRIPTION

Provided herein are methods of determining, assessing, and/or measuringa risk, probability, and/or likelihood of toxicity followingadministration of a therapy, e.g., a cell therapy, that include one ormore steps for measuring, assessing, determining, and/or quantifying afold change in one or more factors indicative of disease burden at twotime points prior to treatment with the therapy, wherein the fold changeindicates the risk, probability, or likelihood of the subject developinga toxicity following administration of and/or associated with thetherapy. Also provided herein are methods of treatment that include oneor more steps of administering to a subject a dose of a therapy, e.g. acell therapy, for treatment of a disease or condition following and/orbased on the results of assessing the fold change in the factorindicative of disease burden, wherein the fold change, when compared toa threshold value, indicates the risk, probability, or likelihood of thesubject developing a toxicity following administration of and/orassociated with the therapy, e.g. cell therapy.

In particular embodiments, the fold change in the factor indicative ofdisease burden is associated with and/or correlated to a risk,probability, and/or likelihood of developing and/or experiencingtoxicity following administration of and/or associated with the therapy.In some embodiments, the factor indicative of disease burden is assessedin subject that is a candidate for receiving the therapy at two timepoints prior to administration of the therapy, e.g., cell therapy.

Immunotherapies, such as adoptive cell therapies (including thoseinvolving the administration of cells expressing chimeric receptorsspecific for a disease or disorder of interest, such as chimeric antigenreceptors (CARs) and/or other recombinant antigen receptors), can beeffective in treating cancer and other diseases and disorders. Incertain contexts, available approaches to adoptive cell therapy may notalways be entirely satisfactory. In some aspects, the providedembodiments are based on observations that the efficacy of adoptive celltherapy may be limited in some context by the development of, or risk ofdeveloping, toxicity or one or more toxic outcomes in the subject. Insome cases, such toxicities can be severe. For example, in some cases,administering a dose of cells expressing a recombinant receptor, e.g. aCAR, can result in toxicity or risk thereof, such as CRS orneurotoxicity.

Predicting the risks of adverse outcomes or toxicity associated with theadministration of a therapy, e.g., a cell therapy, in individualsubjects is useful for evaluating, monitoring, and/or tailoring currentor potential therapies for individual subjects, particularly in thecontext of minimizing potential life-threatening side effects (e.g.,cytokine release syndrome). In some aspects, the methods provided hereinmay be used to minimize, mitigate, and/or avoid the risk of toxicity andother such undesirable outcomes of a therapy, e.g., a cell therapy orCAR-T cell therapy.

The provided methods are based on observations that a fold change in afactor indicative of disease burden, such as measure of tumor volume orlevels or amounts of one or more inflammatory marker, in a subject priorto receiving a therapy, such as a cell therapy (e.g. CAR-T cell therapy)is associated with and/or correlates with a risk of developing toxicityfollowing administration of the therapy. In some embodiments, themethods provided herein include one or more steps of determining a foldchange in a factor indicative of disease burden, by assessing one ormore factors indicative of disease burden, such as measure of tumorvolume or levels or amounts of one or more inflammatory marker, at twoor more different time points prior to administration of the therapy. Insome aspects, the methods can be used to predict or assess the risk thata subject is at risk for developing toxicity following administration ofa therapy in subjects. In some embodiments, the fold change in thefactor indicative of disease burden over two time points is useful forpredicting likelihood of a toxicity developing, for example CRS. In someembodiments, the fold change in the factor indicative of disease burdenover two time points is useful for predicting likelihood of an earlytoxicity developing, for example early CRS that develops within 3, 4, 5,6, or 7 days of administration of the therapy. In particularembodiments, the therapy is a cell therapy, such as a CAR-T celltherapy.

In certain embodiments, the one or more factors indicative of diseaseburden is a volumetric measure of the tumor(s). Examples of a volumetricmeasure of tumor(s) includes a sum of the products of diameters (SPD),longest tumor diameters (LD), sum of longest tumor diameters (SLD),tumor volume, necrosis volume, necrosis-tumor ratio (NTR), peritumoraledema (PTE), and edema-tumor ratio (ETR). In some embodiments, the oneor more factors indicative of disease burden is an inflammatory marker.Examples of inflammatory markers include, but are not limited to,C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), albumin,ferritin, β2 microglobulin (β2-M), or lactate dehydrogenase (LDH).

In some embodiments, the factor indicative of tumor burden is assessedat two time points, a fold change of the factor indicative of diseaseburden between two time points is determined, and the fold change iscompared to a threshold fold change value. In some cases, the foldchange is used to evaluate risk of developing a toxicity followingadministration of a therapy based on whether the fold change is above orbelow a threshold fold change value for the specific factor indicativeof disease burden. In some embodiments, the two time points comprises afirst time point and a second time point, and wherein the fold change isa ratio of the factor indicative of disease burden at the first timepoint and the factor indicative of disease burden at the second timepoint. In some cases, if the cell therapy is to be administered to thesubject, the two time points are both no more than one month or twomonths prior to the subject receiving the cell therapy.

In some embodiments, an advantage of the methods provided herein is thata subject at risk for toxicity may be identified before the therapy isadministered, so that the subject may be assigned close monitoringduring and following treatment of the therapy, receive treatment of thetherapy in a hospital setting, and/or in some embodiments may receive anintervention that prevents, treats, and/or ameliorates toxicity prior toreceiving the therapy or before any symptoms of a toxicity are present.In certain embodiments, the at-risk subject may be selected to receive alow dose of the therapy, or in some cases, to receive an alternativetherapy.

In certain embodiments, the risk, probability, and/or likelihood oftoxicity following administration of or associated with the therapy isassessed in a subject with minimal invasiveness by utilizing imaging orby testing samples, such as serum samples, that are performed orcollected at a screening session, such as a routine assessment or blooddraw to confirm and/or identify the condition or disease in the subject.For example, in some embodiments, the imaging is performed or the sampleis collected at an initial screening, such as when a treatment regimenis still being planned or considered. Thus, in some embodiments, themethods provided herein allow for the risk of toxicity followingadministration of a therapy to be assessed in a subject without the needfor additional procedures, e.g., additional biopsies, beyond the normalprocedures leading up to the therapy.

Unless defined otherwise, all terms of art, notations and othertechnical and scientific terms or terminology used herein are intendedto have the same meaning as is commonly understood by one of ordinaryskill in the art to which the claimed subject matter pertains. In somecases, terms with commonly understood meanings are defined herein forclarity and/or for ready reference, and the inclusion of suchdefinitions herein should not necessarily be construed to represent asubstantial difference over what is generally understood in the art.

All publications, including patent documents, scientific articles anddatabases, referred to in this application are incorporated by referencein their entirety for all purposes to the same extent as if eachindividual publication were individually incorporated by reference. If adefinition set forth herein is contrary to or otherwise inconsistentwith a definition set forth in the patents, applications, publishedapplications and other publications that are herein incorporated byreference, the definition set forth herein prevails over the definitionthat is incorporated herein by reference.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

I. METHODS OF ASSESSING RISK OF TOXICITY OR TOXICITY-RELATED OUTCOMES

Provided herein are methods to assess, predict, infer, and/or estimate arisk of toxicity, for example to a therapy such as a cell therapy, e.g.CAR-T cell therapy. In some embodiments, the subject is administered,will be administered, or is a candidate to be administered a therapy,e.g., an immunotherapy and/or a cell therapy. In some embodiments, thetoxicity is a toxicity following administration of the therapeutictreatment. In certain embodiments, the toxicity is caused by and/orassociated with the therapy. In particular embodiments, methods for theassessment, prediction, inference, and/or estimate of the risk orprobability include one or more steps for measuring or assessing one ormore factors indicative of disease burden at two or more time pointsprior to administration of the therapy. In some embodiments, the methodof assessing a risk of toxicity or toxicity-related outcome includesdetermining a fold change in the factor indicative of disease burdenbetween the two time points. In certain embodiments, the fold change inthe factor indicative of disease burden is predictive of, correlatedwith, and/or associated with a risk of toxicity following administrationof therapy, e.g., the cell therapy. In certain embodiments, the factorindicative of disease burden is a volumetric measure of a tumor. In someembodiments, the factor indicative of disease burden is an inflammatorymarker. In some aspects, the inflammatory marker is measured in asample, e.g., a sample taken, collected, and/or obtained from thesubject.

In some embodiments, the fold change in the factor indicative of diseaseburden, e.g., volumetric measure of the tumor or inflammatory marker, ispredictive of, correlated with, and/or associated with a risk oftoxicity, e.g., toxicity following administration of or associated witha therapy. In certain embodiments the toxicity is to and/or associatedwith a cell therapy. In certain embodiments, the fold change in thefactor indicative of disease burden is indicative of risk of the subjectdeveloping an early toxicity or toxicity-related outcome, e.g., toxicitythat develops within 3, 4, 5, 6, 7, or 8 days after administration ofthe cells therapy. In some embodiments, the toxicity is one or moreoutcomes associated with or related to cytokine release syndrome (CRS),including risk of developing severe CRS or grade 3 or higher CRS. Insome embodiments, the toxicity is one or more outcomes that may be earlyindicators of development of neurotoxicity, such as risk of developingsevere neurotoxicity or grade 3 or higher neurotoxicity.

In particular embodiments, the methods to assess or predict a risk oftoxicity or a toxicity-related outcome, for example, to a therapy suchas a cell therapy, include assessing a factor indicative of diseaseburden at two or more time points prior to receiving a cell therapy in asubject. In some cases, the subject receives a lymphodepleting agentprior to receiving the cell therapy, such as described in Section II,subsection E. In some embodiments, the factor is assessed at twodifferent time points prior to when the therapy, e.g., cell therapy, isadministered. In some cases, both time points are prior toadministration of the lymphodepleting agent. In some embodiments, thetwo time points are both no more than one month or two months prior towhen the therapy is administered. In some embodiments, the two timepoints are not less than one week, two weeks, three weeks, four weeks,or five weeks apart. In some cases, the two time points are not morethan four weeks apart, five weeks, or six weeks apart. In someembodiments, the assessment of the factor indicative of tumor burden atthe first time point is at a screening session, such as a routineassessment or blood draw to confirm and/or identify the condition ordisease in the subject. For example, in some cases, the first time pointis 3 weeks prior to the administration of the cell therapy. In someparticular embodiments, the second time point is more than 1, 2, 3, 4,5, 6, or 7 days prior to administration of the cell therapy. In someparticular embodiments, if the cell therapy is to be administered to thesubject, the second time point is more than 1, 2, 3, 4, 5, 6, or 7 daysbefore administration of the cell therapy. In some embodiments, thesecond time point is prior to the administration of the lymphodepletingagent. In some cases, for example, the first time point is about threeweeks prior to when the therapy is administered and the second timepoint is about 3 days prior to when the therapy is administered. In someembodiments, the method includes determining a fold change in the factorindicative of disease burden between the two time points.

In particular embodiments, provided herein are factors indicative oftumor burden correlated to and/or associated with a risk of developing atoxicity following administration of a therapy, e.g., a cell therapy.For example, in some embodiments, the factor indicative of tumor burdenis a volumetric measure of the tumor(s). Examples of a volumetricmeasure of tumor(s) include a sum of the products of diameters (SPD),longest tumor diameters (LD), sum of longest tumor diameters (SLD),tumor volume, necrosis volume, necrosis-tumor ratio (NTR), peritumoraledema (PTE), and edema-tumor ratio (ETR). In some embodiments, themethods to assess, predict, infer, and/or estimate a risk of toxicity,include one or more steps for using a procedure such as imaging todetermine tumor or tumor-related size or volume. In some cases, thefactor indicative of tumor burden is an inflammatory marker, such asC-reactive protein (CRP), erythrocyte sedimentation rate (ESR), albumin,ferritin, β2 microglobulin (β2-M), or lactate dehydrogenase (LDH). Inparticular embodiments, the methods to assess, predict, infer, and/orestimate a risk of toxicity, include one or more steps for taking,collecting, and/or obtaining a sample from a subject; detecting,measuring, and/or obtaining a presence or level of the factor in thesample. In some cases, one or more of the methods to assess a factorindicative of disease burden can be used.

In some embodiments, the method for assaying toxicity or toxicityrelated outcome includes one or more steps including comparing the foldchange in the factor indicative of tumor burden to a threshold foldchange value for the factor. In certain embodiments, comparing the foldchange in the factor indicative of tumor burden between the two timepoints with the threshold fold change value indicates the risk,probability, and/or likelihood that a subject will experience a toxicityfollowing administration of the therapy if the fold change is above orbelow the threshold fold change value. In provided embodiments, thesubject is or is likely at risk of developing toxicity if the foldchange is at or above the threshold value. In provided embodiments, thesubject is not or is likely not at risk of developing toxicity if thefold change is below the threshold value. In some embodiments, thesample is obtained from a subject that is administered, will beadministered, or is a candidate to be administered a therapy, e.g., animmunotherapy and/or a cell therapy.

In some examples, the threshold value is a value that is within 25%,within 20%, within 15%, within 10%, or within 5% above the average foldchange of the factor indicative of disease burden and/or is within astandard deviation above the average fold change of the factorindicative of disease burden in a plurality of control subjects. In someexamples, the threshold value is above the highest fold change of thefactor indicative of disease burden, such as within 50%, within 25%,within 20%, within 15%, within 10%, or within 5% above such highest foldchange, measured in at least one subject from among a plurality ofcontrol subjects. In some examples, the threshold value is above thehighest fold change as measured among more than 75%, 80%, 85%, 90%, or95%, or 98% of subjects from a plurality of control subjects. In somecases, the plurality of control subjects are a group of subjects priorto receiving a cell therapy for treating a disease or condition, saidcell therapy containing cells genetically engineered to express arecombinant receptor, wherein each of the subjects of the group went onto develop toxicity. In some cases, the plurality of control subjects isa group of subjects having been administered a cell therapy for treatinga disease or condition and having had the fold change of the factorindicative of disease burden assessed, wherein each of the subjects ofthe group went on to develop toxicity, optionally early toxicity,optionally a fever or a sustained fever, within 7 days after receivingthe cell therapy for treating the same disease or condition. In somecases, the plurality of control subjects went on to develop a toxicitythat was detected or observed early or relatively early followingadministration of a cell therapy, such as by a fever or a sustainedfever, within 7 days after receiving the cell therapy for treating thesame disease or condition. In some aspects, the results of the assessingcan inform or determine, such as in some cases alter, the treatmentregimen the subject is to receive in connection with administration ofthe cell therapy.

In some cases, if the assessing indicates the subject is or is likely todevelop toxicity following administration of the cell therapy, thesubject may receive a therapeutic regimen that includes an agent orother treatment capable of treating, preventing, delaying, reducing orattenuating the development or risk of development of a toxicity and thecell therapy. In some embodiments, such an agent or other treatment canbe administered (i) prior to, (ii) within one, two, or three days of,(iii) concurrently with and/or (iv) at first fever following, theinitiation of administration of the cell therapy to the subject. In someaspects, if the assessing indicates the subject is or is likely todevelop toxicity following administration of the cell therapy, the celltherapy is administered at a reduced dose or at a dose that is notassociated with risk of developing toxicity or severe toxicity, or isnot associated with a risk of developing a toxicity or severe toxicityin a majority of subjects, and/or a majority of subjects having adisease or condition that the subject has or is suspected of having,following administration of the cell therapy. In such examples, thereduced dose or dose that is not associated with the risk of toxicitycan be empirically determined, such as based on clinical studies ofsimilarly situated patients. In some embodiments, if the assessingindicates the subject is or is likely to develop a toxicity followingadministration of the cell therapy, the cell therapy can be administeredin an in-patient setting and/or with admission to the hospital for oneor more days. In such examples, the cell therapy may be one thatotherwise is typically to be administered to subjects on an outpatientbasis or without admission to the hospital for one or more days. In somecases, if the assessing indicates the subject is or is likely to developa toxicity following administration of the cell therapy, the subject mayreceive an alternative therapeutic treatment other than the celltherapy. In some of any such embodiments, if the assessing indicates thesubject is or is likely to develop toxicity following administration ofthe cell therapy, the cell therapy is not administered to the subject.It is within the level of a skilled practitioner or physician todetermine the appropriate regimen depending, for example, on the age orother patient specific factors of the subject being treated, the state,course or severity of the disease, the degree of likelihood the subjectmay or likely may develop toxicity, or the likelihood or suspectedlikelihood of one or more other adverse side effects of a therapy orregimen.

In some of any such embodiments, if the assessing indicates the subjectis not or is likely not to develop toxicity following administration ofthe cell therapy, subject may receive the cell therapy, such as at itsstandard dose as determined for treatment of the disease or condition.In some cases, the dose is a non-reduced dose compared to the doseadministered to a plurality of similarly situated subjects that have notgone on to develop a toxicity following administration of the same orsimilar cell therapy. In some cases, the dose is given to the subject onan outpatient basis or without admission to the hospital for one or moredays. In some examples, if the assessing indicates the subject is not oris likely not to develop toxicity following administration of the celltherapy, the subject may receive the cell therapy without administering,prior to or concurrently with administering the cell therapy and/orprior to the development of a sign or symptom of toxicity other thanfever, an agent or treatment capable of treating, preventing, delaying,or attenuating the development of the toxicity. In some cases, if theassessing indicates the subject is not or is likely not to developtoxicity following administration of the cell therapy, the subject mayreceive administration of the cell therapy in an outpatient settingand/or without admission of the subject to the hospital overnight or forone or more days, such as one or more consecutive days. It is within thelevel of a skilled practitioner or physician to determine theappropriate regimen depending, for example, on the age or other patientspecific factors of the subject being treated, the state, course orseverity of the disease, the degree of likelihood the subject may orlikely may develop toxicity, or the likelihood or suspected likelihoodof one or more other adverse side effects of a therapy or regimen.

A. Factors Indicative of Tumor Burden

1. Volumetric Measures of Tumor

Provided herein are methods for detecting and assessing a factorindicative of tumor burden at two time points and determining a foldchange in the factor indicative of tumor burden between the two timepoints. In some embodiments, the factor indicative of tumor burden is avolumetric measure of tumor. In some embodiments, instructions areprovided for assessing tumor or tumor related volume or size in asubject that is a candidate for treatment, optionally with a celltherapy, said cell therapy optionally including a dose or composition ofgenetically engineered cells expressing a recombinant receptor. Alsoprovided is an article of manufacture containing a cell therapycontaining a dose or composition of genetically engineered cellsexpressing a recombinant receptor, and instructions for administeringthe cell therapy following or based on the results of a volumetricassessment of tumor burden in the subject at two time points. In someembodiments, the volumetric measurement of the tumor in the subject isat two time points prior to administering the cell therapy. In someembodiments, the inflammatory marker is a sum of the products ofdiameters (SPD), longest tumor diameters (LD), sum of longest tumordiameters (SLD), necrosis, tumor volume, necrosis volume, necrosis-tumorratio (NTR), peritumoral edema (PTE), and edema-tumor ratio (ETR).

Also provided are methods for selecting a subject for treatmentincluding assessing a volumetric measure of tumor in the subject. Insome embodiments, the fold change in volumetric measure of tumor(s)between the two points is determined and compared to a threshold foldchange value to assess risk of developing a toxicity or atoxicity-related outcome after administering the cell therapy. In someembodiments, the threshold fold change for SPD is about 5 fold, 6 fold,7 fold, 8 fold, or 9 fold. In some embodiments, the threshold value is athreshold value for fold change of a volumetric measure of diseaseburden that is SPD, wherein the fold change is at least about or isabout or is 5 fold, 6 fold, 7 fold, 8 fold, or 9 fold.

a. Methods for Measuring

Provided are methods of detecting and assessing one or more factorsindicative of tumor burden. In some embodiments, the factor indicativeof tumor burden is a volumetric measure of tumor(s). In someembodiments, the volumetric measure is a measure of the lesion(s), suchas the tumor size, tumor diameter, tumor volume, tumor mass, tumor loador bulk, tumor-related edema, tumor-related necrosis, and/or number orextent of metastases. In some embodiments, the volumetric measure oftumor is a bidimensional measure. For example, in some embodiments, thearea of lesion(s) are calculated as the product of the longest diameterand the longest perpendicular diameter of all measurable tumors. In somecases, the volumetric measure of tumor is a unidimensional measure. Insome cases, the size of measurable lesions is assessed as the longestdiameter. In some embodiments, the sum of the products of diameters(SPD), longest tumor diameters (LD), sum of longest tumor diameters(SLD), necrosis, tumor volume, necrosis volume, necrosis-tumor ratio(NTR), peritumoral edema (PTE), and edema-tumor ratio (ETR) is measured.

In some embodiments, instructions are also provided for assessing and/ormaking volumetric measures of tumor in a subject that is a candidate fortreatment, optionally with a cell therapy, said cell therapy optionallyincluding a dose or composition of genetically engineered cellsexpressing a recombinant receptor. For methods for measuring andassessing tumor burden, see Carceller et al., Pediatr Blood Cancer.(2016) 63(8):1400-1406 and Eisenhauer et al., Eur J Cancer. (2009)45(2):228-247. In some embodiments, the volumetric is a sum of theproducts of diameters (SPD) measured by determining the sum of theproducts of the largest perpendicular diameters of all measurabletumors. In some aspects, the tumor or lesion are measured in onedimension with the longest diameter (LD) and/or by determining the sumof longest tumor diameters (SLD) of all measurable lesions. In someembodiments, the volumetric measure of tumor is a volumetricquantification of tumor necrosis, such as necrosis volume and/ornecrosis-tumor ratio (NTR), see Monsky et al., Anticancer Res. (2012)32(11): 4951-4961. In some aspects, the volumetric measure of tumor is avolumetric quantification of tumor-related edema, such as peritumoraledema (PTE) and/or edema-tumor ratio (ETR). In some embodiments,measuring can be performed using imaging techniques such as computedtomography (CT), positron emission tomography (PET), and/or magneticresonance imaging (MRI) of the subject.

In some embodiments, the volumetric measure of tumor is determined attwo time points prior to the administration of the therapy, e.g., celltherapy. In some embodiments, the volumetric measure of tumor isdetermined at a screening session, such as a routine assessment or blooddraw to confirm and/or identify the condition or disease in the subject.

b. Subject

In certain embodiments, volumetric measurement of tumor is obtained fromthe subject at two or more time points to determine a fold change in thefactor indicative of disease burden. In particular embodiments, thesubject has a disease or condition and/or is suspected of having adisease or condition. In some embodiments, subject has received, willreceive, or is a candidate to receive a therapy. In some embodiments,the therapy is an administration of a cell therapy. In particularembodiments, the therapy is an immunotherapy, e.g., cell therapy. Incertain embodiments, the cell therapy treats and/or is capable oftreating the disease or condition. In some embodiments, the therapy is acell therapy that contains one or more engineered cells. In someembodiments, the engineered cells express a recombinant receptor. Inparticular embodiments, the recombinant receptor is a CAR. In particularembodiments, the sample is taken, collected, and/or obtained from asubject who has been, who will be, or is a candidate to be administereda therapy. In particular embodiments, the sample is taken, collected,and/or obtained prior to treatment or administration with the therapy,e.g., the cell therapy.

In particular embodiments, the volumetric measurement of the tumor(s) isdetermined a subject who has been, who will be, or is a candidate to beadministered a therapy. In particular embodiments, the measurement isdetermined prior to treatment or administration with the therapy, e.g.,the cell therapy. In accord with methods, kits and articles ofmanufacture described herein, the volumetric measure of tumor isassociated with and/or correlate to toxicity or risk of toxicity.Exemplary volumetric measures associated with and/or correlated with arisk of developing toxicity that may be detected in a sample collectedor obtained from a subject prior to receiving an immunotherapy includesum of the products of diameters (SPD), longest tumor diameters (LD),sum of longest tumor diameters (SLD), necrosis, tumor volume, necrosisvolume, necrosis-tumor ratio (NTR), peritumoral edema (PTE), andedema-tumor ratio (ETR). Thus, in some aspects, the provided methodsrelate to identifying subjects, prior to receiving an immunotherapy,such as a cell therapy (e.g. CAR-T cells), who may be at risk ofdeveloping a toxicity, e.g. CRS. As described elsewhere herein, themethods can be used to determine if the subject should be closelymonitored following the administration of the immunotherapy, is acandidate for outpatient therapy or should receive treatment of thetherapy in a hospital setting and/or is a candidate for receiving anintervention of preventing, treating or ameliorating a risk of atoxicity.

In some embodiments, the volumetric measure of a tumor is determined ina subject that has or is suspected of having a condition or disease. Insome embodiments, the subject has or is suspected of having a cancer orproliferative disease. In particular embodiments, the subject has adisease or condition, or is suspected of having a disease or condition,that is associated with an antigen and/or is associated with diseasedcells that express the antigen. In some embodiments, the disease orcondition, e.g., a cancer or proliferative disorder, is associated withαvP6 integrin (avb6 integrin), B cell maturation antigen (BCMA), B7-H6,carbonic anhydrase 9 (CA9, also known as CAIX or G250), a cancer-testisantigen, cancer/testis antigen 1B (CTAG, also known as NY-ESO-1 andLAGE-2), carcinoembryonic antigen (CEA), a cyclin, cyclin A2, C-C MotifChemokine Ligand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33,CD38, CD44, CD44v6, CD44v7/8, CD123, CD138, CD171, epidermal growthfactor protein (EGFR), type III epidermal growth factor receptormutation (EGFR VIII), epithelial glycoprotein 2 (EPG-2), epithelialglycoprotein 40 (EPG-40), ephrinB2, ephrine receptor A2 (EPHa2),estrogen receptor, Fc receptor like 5 (FCRL5; also known as Fc receptorhomolog 5 or FCRH5), fetal acetylcholine receptor (fetal AchR), a folatebinding protein (FBP), folate receptor alpha, fetal acetylcholinereceptor, ganglioside GD2, O-acetylated GD2 (OGD2), ganglioside GD3,glycoprotein 100 (gp100), Her2/neu (receptor tyrosine kinase erbB2),Her3 (erb-B3), Her4 (erb-B4), erbB dimers, human high molecularweight-melanoma-associated antigen (HMW-MAA), hepatitis B surfaceantigen, Human leukocyte antigen A1 (HLA-AI), human leukocyte antigen A2(HLA-A2), IL-22 receptor alpha(IL-22Ra), IL-13 receptor alpha 2(IL-13Ra2), kinase insert domain receptor (kdr), kappa light chain, L1cell adhesion molecule (L1CAM), CE7 epitope of L-CAM, Leucine RichRepeat Containing 8 Family Member A (LRRC8A), Lewis Y,melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, mesothelin,c-Met, murine cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, naturalkiller group 2 member D (NKG2D) ligands, melan A (MART-1), neural celladhesion molecule (NCAM), oncofetal antigen, preferentially expressedantigen of melanoma (PRAME), progesterone receptor, a prostate specificantigen, prostate stem cell antigen (PSCA), prostate specific membraneantigen (PSMA), receptor tyrosine kinase like orphan receptor 1 (ROR1),survivin, Trophoblast glycoprotein (TPBG also known as 5T4),tumor-associated glycoprotein 72 (TAG72), vascular endothelial growthfactor receptor (VEGFR), vascular endothelial growth factor receptor 2(VEGFR2), Wilms tumor 1 (WT-1), and/or a pathogen-specific antigen. Incertain embodiments, the subject has a disease or condition, or issuspected of having a disease or condition, that is associated with CD19and/or is associated with diseased cells that express CD19.

In some embodiments, the disease or condition, e.g., a cancer orproliferative disorder, is associated with αvP6 integrin (avb6integrin), B cell maturation antigen (BCMA), B7-H3, B7-H6, carbonicanhydrase 9 (CA9, also known as CAIX or G250), a cancer-testis antigen,cancer/testis antigen 1B (CTAG, also known as NY-ESO-1 and LAGE-2),carcinoembryonic antigen (CEA), a cyclin, cyclin A2, C-C Motif ChemokineLigand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44,CD44v6, CD44v7/8, CD123, CD133, CD138, CD171, chondroitin sulfateproteoglycan 4 (CSPG4), epidermal growth factor protein (EGFR), type IIIepidermal growth factor receptor mutation (EGFR vIII), epithelialglycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrinB2,ephrin receptor A2 (EPHa2), estrogen receptor, Fc receptor like 5(FCRL5; also known as Fc receptor homolog 5 or FCRH5), fetalacetylcholine receptor (fetal AchR), a folate binding protein (FBP),folate receptor alpha, ganglioside GD2, O-acetylated GD2 (OGD2),ganglioside GD3, glycoprotein 100 (gp100), glypican-3 (GPC3), G ProteinCoupled Receptor 5D (GPRC5D), Her2/neu (receptor tyrosine kinaseerb-B2), Her3 (erb-B3), Her4 (erb-B4), erbB dimers, Human high molecularweight-melanoma-associated antigen (HMW-MAA), hepatitis B surfaceantigen, Human leukocyte antigen A1 (HLA-A1), Human leukocyte antigen A2(HLA-A2), IL-22 receptor alpha(IL-22Rα), IL-13 receptor alpha 2(IL-13Ra2), kinase insert domain receptor (kdr), kappa light chain, L1cell adhesion molecule (L1-CAM), CE7 epitope of L-CAM, Leucine RichRepeat Containing 8 Family Member A (LRRC8A), Lewis Y,Melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MAGE-A10,mesothelin (MSLN), c-Met, murine cytomegalovirus (CMV), mucin 1 (MUC1),MUC16, natural killer group 2 member D (NKG2D) ligands, melan A(MART-1), neural cell adhesion molecule (NCAM), oncofetal antigen,Preferentially expressed antigen of melanoma (PRAME), progesteronereceptor, a prostate specific antigen, prostate stem cell antigen(PSCA), prostate specific membrane antigen (PSMA), Receptor TyrosineKinase Like Orphan Receptor 1 (ROR1), survivin, Trophoblast glycoprotein(TPBG also known as 5T4), tumor-associated glycoprotein 72 (TAG72),Tyrosinase related protein 1 (TRP1, also known as TYRP1 or gp75),Tyrosinase related protein 2 (TRP2, also known as dopachrometautomerase, dopachrome delta-isomerase or DCT), vascular endothelialgrowth factor receptor (VEGFR), vascular endothelial growth factorreceptor 2 (VEGFR2), Wilms Tumor 1 (WT-1), a pathogen-specific orpathogen-expressed antigen, or an antigen associated with a universaltag, and/or biotinylated molecules, and/or molecules expressed by HIV,HCV, HBV or other pathogens. Antigens targeted by the receptors in someembodiments include antigens associated with a B cell malignancy, suchas any of a number of known B cell marker. In some embodiments, theantigen is or includes CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33,Igkappa, Iglambda, CD79a, CD79b or CD30.

In some embodiments, the volumetric measure of tumor is determined in asubject that has or is suspected of having a cancer or proliferativedisease that is a B cell malignancy or hematological malignancy. In someembodiments, the cancer or proliferative disease is a myeloma, e.g., amultiple myeloma (MM), a lymphoma or a leukemia, lymphoblastic leukemia(ALL), non-Hodgkin's lymphoma (NHL), chronic lymphocytic leukemia (CLL),a diffuse large B-cell lymphoma (DLBCL), and/or acute myeloid leukemia(AML). In some embodiments, the cancer or proliferative disorder is ALL.In some embodiments, the subject has, or is suspected of having ALL. Insome embodiments, the ALL is adult ALL. In particular embodiments, theALL is pediatric ALL.

In particular embodiments, the volumetric measure of tumor(s) in thesubject is determined at two or more time points prior to administrationof the therapy. In some embodiments, the volumetric measure of tumor ofthe subject is determined at a screening session, such as a routineassessment to confirm and/or identify the condition or disease in thesubject.

2. Inflammatory Markers

Provided herein are methods for detecting and assessing a factorindicative of tumor burden at two time points and determining a foldchange in the factor indicative of tumor burden between the two timepoints. In some embodiments, the factor indicative of tumor burden is aninflammatory marker. Also provided are articles of manufacturecontaining a reagent capable of detecting or that is specific for aninflammatory marker. In some embodiments, instructions are provided forassessing a biological sample for the inflammatory marker from a subjectthat is a candidate for treatment, optionally with a cell therapy, saidcell therapy optionally including a dose or composition of geneticallyengineered cells expressing a recombinant receptor. Also provided areinstructions for using the reagents to detect the inflammatory markerand assess the one or more inflammatory marker in a sample obtained froma subject that is a candidate for treatment, optionally with a celltherapy.

Also provided is an article of manufacture containing a cell therapycontaining a dose or composition of genetically engineered cellsexpressing a recombinant receptor, and instructions for administeringthe cell therapy following or based on the results of an assessment, ina biological sample of the presence or level of inflammatory marker. Insome embodiments, the biological sample is obtained from the subjectprior to administering the cell therapy. In some embodiments, theinflammatory marker is C-reactive protein (CRP), erythrocytesedimentation rate (ESR), albumin, ferritin, β2 microglobulin (β2-M), orlactate dehydrogenase (LDH). Also provided are methods for selecting asubject for treatment including contacting a biological sample (e.g.blood or serum sample) with the reagent capable of detecting or that isspecific for an inflammatory marker. In some cases, the reagent iscapable of detecting C-reactive protein (CRP), erythrocyte sedimentationrate (ESR), albumin, ferritin, β2 microglobulin (β2-M), or lactatedehydrogenase (LDH).

In some embodiments, the fold change in inflammatory marker(s) betweenthe two points is determined and compared to a threshold fold changevalue to assess risk of developing a toxicity or a toxicity-relatedoutcome after administering the cell therapy.

a. Reagents for Measuring

Provided herein are articles of manufacture and containing a reagentcapable of detecting or that is specific for an inflammatory marker andmethods for using the articles of manufacture. In some embodiments,instructions are also provided for using the reagent to assay abiological sample from a subject that is a candidate for treatment,optionally with a cell therapy, said cell therapy optionally including adose or composition of genetically engineered cells expressing arecombinant receptor. In some embodiments of using the articles ofmanufacture, the level or presence of C-reactive protein (CRP),erythrocyte sedimentation rate (ESR), albumin, ferritin, β2microglobulin (β2-M), or lactate dehydrogenase (LDH) is detected andassessed. Also provided are methods of detecting and assessing one ormore inflammatory markers indicative of tumor burden.

In some embodiments, the inflammatory marker is assessed using an immuneassay. For example, an enzyme-linked immunosorbent assay (ELISA), enzymeimmunoassay (EIA), radioimmunoassay (RIA), surface plasmon resonance(SPR), Western Blot, Lateral flow assay, immunohistochemistry, proteinarray or immuno-PCR (iPCR) can be used to detect the inflammatorymarker. In some embodiments, using the articles of manufacture includedetecting an inflammatory marker indicative of tumor burden. In somecases, the assaying or assessing of an inflammatory marker is using flowcytometry. In some cases, the reagent is a soluble protein that bindsthe inflammatory marker. In some example, the reagent is a protein thatbinds C-reactive protein (CRP), erythrocyte sedimentation rate (ESR),albumin, ferritin, 2 microglobulin (β2-M), or lactate dehydrogenase(LDH).

In some embodiments, C-reactive protein (CRP) is assessed using an invitro enzyme-linked immunosorbent assay to obtain a quantitativemeasurement of human CRP from a sample such as serum, plasma, or blood.In some examples, CRP is detected using a human Enzyme-LinkedImmunosorbent Assay (ELISA). In some embodiments, erythrocytesedimentation rate (ESR) is assessed by measuring the distance (inmillimeters per hour) that red cells have fallen after separating fromthe plasma in a vertical pipette or tube. In some aspects, albumin isassessed using a colorimetric test or an in vitro enzyme-linkedimmunosorbent assay. In some examples, albumin is detected using a humanEnzyme-Linked Immunosorbent Assay (ELISA). In some embodiments, ferritinor β2 microglobulin is assessed using an immunoassay or detected usingan ELISA. In some aspects, lactate dehydrogenase (LDH) is assessed usinga colorimetric test or an in vitro enzyme-linked immunosorbent assay.

The term “antibody” herein is used in the broadest sense and includespolyclonal and monoclonal antibodies, including intact antibodies andfunctional (antigen-binding) antibody fragments, including fragmentantigen binding (Fab) fragments, F(ab′)₂ fragments, Fab′ fragments, Fvfragments, recombinant IgG (rIgG) fragments, single chain antibodyfragments, including single chain variable fragments (scFv), and singledomain antibodies (e.g., sdAb, sdFv, nanobody) fragments. The termencompasses genetically engineered and/or otherwise modified forms ofimmunoglobulins, such as intrabodies, peptibodies, chimeric antibodies,fully human antibodies, humanized antibodies, and heteroconjugateantibodies, multispecific, e.g., bispecific, antibodies, diabodies,triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv. Unlessotherwise stated, the term “antibody” should be understood to encompassfunctional antibody fragments thereof. The term also encompasses intactor full-length antibodies, including antibodies of any class orsub-class, including IgG and sub-classes thereof, IgM, IgE, IgA, andIgD.

The terms “complementarity determining region,” and “CDR,” synonymouswith “hypervariable region” or “HVR,” are known in the art to refer tonon-contiguous sequences of amino acids within antibody variableregions, which confer antigen specificity and/or binding affinity. Ingeneral, there are three CDRs in each heavy chain variable region(CDR-H1, CDR-H2, CDR-H3) and three CDRs in each light chain variableregion (CDR-L1, CDR-L2, CDR-L3). “Framework regions” and “FR” are knownin the art to refer to the non-CDR portions of the variable regions ofthe heavy and light chains. In general, there are four FRs in eachfull-length heavy chain variable region (FR-H1, FR-H2, FR-H3, andFR-H4), and four FRs in each full-length light chain variable region(FR-L1, FR-L2, FR-L3, and FR-L4).

The precise amino acid sequence boundaries of a given CDR or FR can bereadily determined using any of a number of well-known schemes,including those described by Kabat et al. (1991), “Sequences of Proteinsof Immunological Interest,” 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (“Kabat” numbering scheme);Al-Lazikani et al., (1997) JMB 273, 927-948 (“Chothia” numberingscheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996),“Antibody-antigen interactions: Contact analysis and binding sitetopography,” J. Mol. Biol. 262, 732-745.” (“Contact” numbering scheme);Lefranc M P et al., “IMGT unique numbering for immunoglobulin and T cellreceptor variable domains and Ig superfamily V-like domains,” Dev CompImmunol, 2003 January; 27(1):55-77 (“IMGT” numbering scheme); Honegger Aand Pluckthun A, “Yet another numbering scheme for immunoglobulinvariable domains: an automatic modeling and analysis tool,” J Mol Biol,2001 Jun. 8; 309(3):657-70, (“Aho” numbering scheme); and Martin et al.,“Modeling antibody hypervariable loops: a combined algorithm,” PNAS,1989, 86(23):9268-9272, (“AbM” numbering scheme).

The boundaries of a given CDR or FR may vary depending on the schemeused for identification. For example, the Kabat scheme is based onstructural alignments, while the Chothia scheme is based on structuralinformation. Numbering for both the Kabat and Chothia schemes is basedupon the most common antibody region sequence lengths, with insertionsaccommodated by insertion letters, for example, “30a,” and deletionsappearing in some antibodies. The two schemes place certain insertionsand deletions (“indels”) at different positions, resulting indifferential numbering. The Contact scheme is based on analysis ofcomplex crystal structures and is similar in many respects to theChothia numbering scheme. The AbM scheme is a compromise between Kabatand Chothia definitions based on that used by Oxford Molecular's AbMantibody modeling software.

Table 1A, below, lists exemplary position boundaries of CDR-L1, CDR-L2,CDR-L3 and CDR-H1, CDR-H2, CDR-H3 as identified by Kabat, Chothia, AbM,and Contact schemes, respectively. For CDR-H1, residue numbering islisted using both the Kabat and Chothia numbering schemes. FRs arelocated between CDRs, for example, with FR-L1 located before CDR-L1,FR-L2 located between CDR-L1 and CDR-L2, FR-L3 located between CDR-L2and CDR-L3 and so forth. It is noted that because the shown Kabatnumbering scheme places insertions at H35A and H35B, the end of theChothia CDR-H1 loop when numbered using the shown Kabat numberingconvention varies between H32 and H34, depending on the length of theloop.

TABLE 1A Boundaries of CDRs according to various numbering schemes. CDRKabat Chothia AbM Contact CDR-L1 L24--L34 L24--L34 L24--L34 L30--L36CDR-L2 L50--L56 L50--L56 L50--L56 L46--L55 CDR-L3 L89--L97 L89--L97L89--L97 L89--L96 CDR-H1 H31--H35B H26--H32.34 H26--H35B H30--H35B(Kabat Numbering¹) CDR-H1 H31--H35 H26--H32 H26--H35 H30--H35 (ChothiaNumbering²) CDR-H2 H50--H65 H52--H56 H50--H58 H47--H58 CDR-H3 H95--H102H95--H102 H95--H102 H93--H101 ¹Kabat et al. (1991), “Sequences ofProteins of Immunological Interest,” 5th Ed. Public Health Service,National Institutes of Health, Bethesda, MD ²Al-Lazikani et al., (1997)JMB 273, 927-948

Thus, unless otherwise specified, a “CDR” or “complementary determiningregion,” or individual specified CDRs (e.g., CDR-H1, CDR-H2, CDR-H3), ofa given antibody or region thereof, such as a variable region thereof,should be understood to encompass a (or the specific) complementarydetermining region as defined by any of the aforementioned schemes, orother known schemes. For example, where it is stated that a particularCDR (e.g., a CDR-H3) contains the amino acid sequence of a correspondingCDR in a given V_(H) or V_(L) region amino acid sequence, it isunderstood that such a CDR has a sequence of the corresponding CDR(e.g., CDR-H3) within the variable region, as defined by any of theaforementioned schemes, or other known schemes. In some embodiments,specific CDR sequences are specified. Exemplary CDR sequences ofprovided antibodies are described using various numbering schemes,although it is understood that a provided antibody can include CDRs asdescribed according to any of the other aforementioned numbering schemesor other numbering schemes known to a skilled artisan.

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable regions of the heavy chain and lightchain (V_(H) and V_(L), respectively) of a native antibody generallyhave similar structures, with each domain comprising four conservedframework regions (FRs) and three CDRs. (See, e.g., Kindt et al. KubyImmunology, 6th ed., W.H. Freeman and Co., page 91 (2007). A singleV_(H) or V_(L) domain may be sufficient to confer antigen-bindingspecificity. Furthermore, antibodies that bind a particular antigen maybe isolated using a V_(H) or V_(L) domain from an antibody that bindsthe antigen to screen a library of complementary V_(L) or V_(H) domains,respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887(1993); Clarkson et al., Nature 352:624-628 (1991).

Among the antibodies included in the provided CARs are antibodyfragments. An “antibody fragment” or “antigen-binding fragment” refersto a molecule other than an intact antibody that comprises a portion ofan intact antibody that binds the antigen to which the intact antibodybinds. Examples of antibody fragments include but are not limited to Fv,Fab, Fab′, Fab′-SH, F(ab′)₂; diabodies; linear antibodies; heavy chainvariable (V_(H)) regions, single-chain antibody molecules such as scFvsand single-domain antibodies comprising only the V_(H) region; andmultispecific antibodies formed from antibody fragments. In someembodiments, the antigen-binding domain in the provided CARs is orcomprises an antibody fragment comprising a variable heavy chain (V_(H))and a variable light chain (V_(L)) region. In particular embodiments,the antibodies are single-chain antibody fragments comprising a heavychain variable (V_(H)) region and/or a light chain variable (V_(L))region, such as scFvs.

Among the provided antibodies are antibody fragments. An “antibodyfragment” refers to a molecule other than an intact antibody thatcomprises a portion of an intact antibody that binds the antigen towhich the intact antibody binds. Examples of antibody fragments includebut are not limited to Fv, Fab, Fab′, Fab′-SH, F(ab′)₂; diabodies;linear antibodies; single-chain antibody molecules (e.g. scFv); andmultispecific antibodies formed from antibody fragments. In particularembodiments, the antibodies are single-chain antibody fragmentscomprising a variable heavy chain region and/or a variable light chainregion, such as scFvs.

Single-domain antibodies are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody.

Antibody fragments can be made by various techniques, including but notlimited to proteolytic digestion of an intact antibody as well asproduction by recombinant host cells. In some embodiments, theantibodies are recombinantly produced fragments, such as fragmentscomprising arrangements that do not occur naturally, such as those withtwo or more antibody regions or chains joined by synthetic linkers,e.g., peptide linkers, and/or that are may not be produced by enzymedigestion of a naturally-occurring intact antibody. In some aspects, theantibody fragments are scFvs.

A “humanized” antibody is an antibody in which all or substantially allCDR amino acid residues are derived from non-human CDRs and all orsubstantially all FR amino acid residues are derived from human FRs. Ahumanized antibody optionally may include at least a portion of anantibody constant region derived from a human antibody. A “humanizedform” of a non-human antibody, refers to a variant of the non-humanantibody that has undergone humanization, typically to reduceimmunogenicity to humans, while retaining the specificity and affinityof the parental non-human antibody. In some embodiments, some FRresidues in a humanized antibody are substituted with correspondingresidues from a non-human antibody (e.g., the antibody from which theCDR residues are derived), e.g., to restore or improve antibodyspecificity or affinity.

Among the provided antibodies are human antibodies. A “human antibody”is an antibody with an amino acid sequence corresponding to that of anantibody produced by a human or a human cell, or non-human source thatutilizes human antibody repertoires or other human antibody-encodingsequences, including human antibody libraries. The term excludeshumanized forms of non-human antibodies comprising non-humanantigen-binding regions, such as those in which all or substantially allCDRs are non-human.

Human antibodies may be prepared by administering an immunogen to atransgenic animal that has been modified to produce intact humanantibodies or intact antibodies with human variable regions in responseto antigenic challenge. Such animals typically contain all or a portionof the human immunoglobulin loci, which replace the endogenousimmunoglobulin loci, or which are present extrachromosomally orintegrated randomly into the animal's chromosomes. In such transgenicanimals, the endogenous immunoglobulin loci have generally beeninactivated. Human antibodies also may be derived from human antibodylibraries, including phage display and cell-free libraries, containingantibody-encoding sequences derived from a human repertoire.

Among the provided antibodies are monoclonal antibodies, includingmonoclonal antibody fragments. The term “monoclonal antibody” as usedherein refers to an antibody obtained from or within a population ofsubstantially homogeneous antibodies, i.e., the individual antibodiescomprising the population are identical, except for possible variantscontaining naturally occurring mutations or arising during production ofa monoclonal antibody preparation, such variants generally being presentin minor amounts. In contrast to polyclonal antibody preparations, whichtypically include different antibodies directed against differentepitopes, each monoclonal antibody of a monoclonal antibody preparationis directed against a single epitope on an antigen. The term is not tobe construed as requiring production of the antibody by any particularmethod. A monoclonal antibody may be made by a variety of techniques,including but not limited to generation from a hybridoma, recombinantDNA methods, phage-display and other antibody display methods.

Also provided are antibody immunoconjugates comprising an antibodyagainst the marker expressed on a population of myeloid cells attachedto a label, which can generate a detectable signal, indirectly ordirectly. These antibody immunoconjugates can be used for research ordiagnostic applications. The label is preferably capable of producing,either directly or indirectly, a detectable signal. For example, thelabel may be radio-opaque or a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S,¹²³I, ¹²⁵I, ¹³¹I; a fluorescent (fluorophore) or chemiluminescent(chromophore) compound, such as fluorescein isothiocyanate, rhodamine orluciferin; an enzyme, such as alkaline phosphatase, β-galactosidase orhorseradish peroxidase; an imaging agent; or a metal ion. In someembodiments, the label is a radioactive atom for scintigraphic studies,for example ⁹⁹Tc or ¹²³I, or a spin label for nuclear magnetic resonance(NMR) imaging (also known as magnetic resonance imaging, MRI), such aszirconium-89, iodine-123, iodine-131, indium-111, fluorine-19,carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.Zirconium-89 may be complexed to various metal chelating agents andconjugated to antibodies, e.g., for PET imaging (WO 2011/056983).

In some embodiments, the antibody immunoconjugate is detectableindirectly. For example, a secondary antibody that is specific for theantibody against the marker expressed on a population of myeloid cellsimmunoconjugate and contains a detectable label can be used to detectthe antibody immunoconjugate.

In some embodiments, antibodies capable of detecting or that is specificthe inflammatory markers provided herein may be identified, screenedfor, or characterized for their physical/chemical properties and/orbiological activities by various known assays. In one aspect, theantibody is tested for its antigen binding activity, e.g., by knownmethods such as an immunoassay, ELISA, Western blotting, and/or flowcytometric assays, including cell-based binding assays.

b. Samples

In certain embodiments, one or more inflammatory are measured, assessed,and/or determined in one or more samples obtained at two or more timepoints to determine a fold change in the factor indicative of diseaseburden. In particular embodiments, the sample is a biological samplethat is taken, collected, and/or obtained from a subject. In certainembodiments, the subject has a disease or condition and/or is suspectedof having a disease or condition. In some embodiments, subject hasreceived, will receive, or is a candidate to receive a therapy. In someembodiments, the therapy is an administration of a cell therapy. Inparticular embodiments, the therapy is an immunotherapy. In certainembodiments, the cell therapy treats and/or is capable of treating thedisease or condition. In some embodiments, the therapy is a cell therapythat contains one or more engineered cells. In some embodiments, theengineered cells express a recombinant receptor. In particularembodiments, the recombinant receptor is a CAR. In particularembodiments, the sample is taken, collected, and/or obtained from asubject who has been, who will be, or is a candidate to be administereda therapy. In particular embodiments, the sample is taken, collected,and/or obtained prior to treatment or administration with the therapy,e.g., the cell therapy.

In particular embodiments, the sample is taken, collected, and/orobtained from a subject who has been, who will be, or is a candidate tobe administered a therapy. In particular embodiments, the sample istaken, collected, and/or obtained prior to treatment or administrationwith the therapy, e.g., the cell therapy. In accord with methods, kitsand articles of manufacture described herein, the sample can be assessedfor one or more inflammatory markers that is associated with and/orcorrelate to toxicity or risk of toxicity. Exemplary inflammatorymarkers associated with and/or correlated with a risk of developingtoxicity that may be detected in a sample collected or obtained from asubject prior to receiving an immunotherapy include C-reactive protein(CRP), erythrocyte sedimentation rate (ESR), albumin, ferritin, β2microglobulin (β2-M), or lactate dehydrogenase (LDH). Thus, in someaspects, the provided methods relate to identifying subjects, prior toreceiving an immunotherapy, such as a cell therapy (e.g. CAR-T cells),who may be at risk of developing a toxicity, e.g. CRS. As describedelsewhere herein, the methods can be used to determine if the subjectshould be closely monitored following the administration of theimmunotherapy, is a candidate for outpatient therapy or should receivetreatment of the therapy in a hospital setting and/or is a candidate forreceiving an intervention of preventing, treating or ameliorating a riskof a toxicity.

In some embodiments, the sample is taken, collected, and/or obtainedfrom a subject that has or is suspected of having a condition ordisease. In some embodiments, the subject has or is suspected of havinga cancer or proliferative disease. In particular embodiments, thesubject has a disease or condition, or is suspected of having a diseaseor condition, that is associated with an antigen and/or is associatedwith diseased cells that express the antigen. In some embodiments, thedisease or condition, e.g., a cancer or proliferative disorder, isassociated with αvP6 integrin (avb6 integrin), B cell maturation antigen(BCMA), B7-H6, carbonic anhydrase 9 (CA9, also known as CAIX or G250), acancer-testis antigen, cancer/testis antigen 1B (CTAG, also known asNY-ESO-1 and LAGE-2), carcinoembryonic antigen (CEA), a cyclin, cyclinA2, C-C Motif Chemokine Ligand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24,CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD123, CD138, CD171, epidermalgrowth factor protein (EGFR), type III epidermal growth factor receptormutation (EGFR vIII), epithelial glycoprotein 2 (EPG-2), epithelialglycoprotein 40 (EPG-40), ephrinB2, ephrine receptor A2 (EPHa2),estrogen receptor, Fc receptor like 5 (FCRL5; also known as Fc receptorhomolog 5 or FCRH5), fetal acetylcholine receptor (fetal AchR), a folatebinding protein (FBP), folate receptor alpha, fetal acetylcholinereceptor, ganglioside GD2, O-acetylated GD2 (OGD2), ganglioside GD3,glycoprotein 100 (gp100), Her2/neu (receptor tyrosine kinase erbB2),Her3 (erb-B3), Her4 (erb-B4), erbB dimers, human high molecularweight-melanoma-associated antigen (HMW-MAA), hepatitis B surfaceantigen, Human leukocyte antigen A1 (HLA-AI), human leukocyte antigen A2(HLA-A2), IL-22 receptor alpha(IL-22Ra), IL-13 receptor alpha 2(IL-13Ra2), kinase insert domain receptor (kdr), kappa light chain, L1cell adhesion molecule (L1CAM), CE7 epitope of L-CAM, Leucine RichRepeat Containing 8 Family Member A (LRRC8A), Lewis Y,melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, mesothelin,c-Met, murine cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, naturalkiller group 2 member D (NKG2D) ligands, melan A (MART-1), neural celladhesion molecule (NCAM), oncofetal antigen, preferentially expressedantigen of melanoma (PRAME), progesterone receptor, a prostate specificantigen, prostate stem cell antigen (PSCA), prostate specific membraneantigen (PSMA), receptor tyrosine kinase like orphan receptor 1 (ROR1),survivin, Trophoblast glycoprotein (TPBG also known as 5T4),tumor-associated glycoprotein 72 (TAG72), vascular endothelial growthfactor receptor (VEGFR), vascular endothelial growth factor receptor 2(VEGFR2), Wilms tumor 1 (WT-1), and/or a pathogen-specific antigen. Incertain embodiments, the subject has a disease or condition, or issuspected of having a disease or condition, that is associated with CD19and/or is associated with diseased cells that express CD19.

In some embodiments, the disease or condition, e.g., a cancer orproliferative disorder, is associated with αvP6 integrin (avb6integrin), B cell maturation antigen (BCMA), B7-H3, B7-H6, carbonicanhydrase 9 (CA9, also known as CAIX or G250), a cancer-testis antigen,cancer/testis antigen 1B (CTAG, also known as NY-ESO-1 and LAGE-2),carcinoembryonic antigen (CEA), a cyclin, cyclin A2, C-C Motif ChemokineLigand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44,CD44v6, CD44v7/8, CD123, CD133, CD138, CD171, chondroitin sulfateproteoglycan 4 (CSPG4), epidermal growth factor protein (EGFR), type IIIepidermal growth factor receptor mutation (EGFR VIII), epithelialglycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrinB2,ephrin receptor A2 (EPHa2), estrogen receptor, Fc receptor like 5(FCRL5; also known as Fc receptor homolog 5 or FCRH5), fetalacetylcholine receptor (fetal AchR), a folate binding protein (FBP),folate receptor alpha, ganglioside GD2, O-acetylated GD2 (OGD2),ganglioside GD3, glycoprotein 100 (gp100), glypican-3 (GPC3), G ProteinCoupled Receptor 5D (GPRC5D), Her2/neu (receptor tyrosine kinaseerb-B2), Her3 (erb-B3), Her4 (erb-B4), erbB dimers, Human high molecularweight-melanoma-associated antigen (HMW-MAA), hepatitis B surfaceantigen, Human leukocyte antigen A1 (HLA-A1), Human leukocyte antigen A2(HLA-A2), IL-22 receptor alpha(IL-22Rα), IL-13 receptor alpha 2(IL-13Ra2), kinase insert domain receptor (kdr), kappa light chain, L1cell adhesion molecule (L1-CAM), CE7 epitope of L-CAM, Leucine RichRepeat Containing 8 Family Member A (LRRC8A), Lewis Y,Melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MAGE-A10,mesothelin (MSLN), c-Met, murine cytomegalovirus (CMV), mucin 1 (MUC1),MUC16, natural killer group 2 member D (NKG2D) ligands, melan A(MART-1), neural cell adhesion molecule (NCAM), oncofetal antigen,Preferentially expressed antigen of melanoma (PRAME), progesteronereceptor, a prostate specific antigen, prostate stem cell antigen(PSCA), prostate specific membrane antigen (PSMA), Receptor TyrosineKinase Like Orphan Receptor 1 (ROR1), survivin, Trophoblast glycoprotein(TPBG also known as 5T4), tumor-associated glycoprotein 72 (TAG72),Tyrosinase related protein 1 (TRP1, also known as TYRP1 or gp75),Tyrosinase related protein 2 (TRP2, also known as dopachrometautomerase, dopachrome delta-isomerase or DCT), vascular endothelialgrowth factor receptor (VEGFR), vascular endothelial growth factorreceptor 2 (VEGFR2), Wilms Tumor 1 (WT-1), a pathogen-specific orpathogen-expressed antigen, or an antigen associated with a universaltag, and/or biotinylated molecules, and/or molecules expressed by HIV,HCV, HBV or other pathogens. Antigens targeted by the receptors in someembodiments include antigens associated with a B cell malignancy, suchas any of a number of known B cell marker. In some embodiments, theantigen is or includes CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33,Igkappa, Iglambda, CD79a, CD79b or CD30.

In some embodiments, the sample is taken, collected, and/or obtainedfrom a subject that has or is suspected of having a cancer orproliferative disease that is a B cell malignancy or hematologicalmalignancy. In some embodiments, the cancer or proliferative disease isa myeloma, e.g., a multiple myeloma (MM), a lymphoma or a leukemia,lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma (NHL), chroniclymphocytic leukemia (CLL), a diffuse large B-cell lymphoma (DLBCL),and/or acute myeloid leukemia (AML). In some embodiments, the cancer orproliferative disorder is ALL. In some embodiments, the subject has, oris suspected of having ALL. In some embodiments, the ALL is adult ALL.In particular embodiments, the ALL is pediatric ALL.

In particular embodiments, two or more samples are obtained, collected,or taken from the subject prior to administration of the therapy. Incertain embodiments, the sample is a biological sample. In certainembodiments, the sample is a blood sample, plasma sample, or serumsample. In certain embodiments, the sample is a tissue sample. In someembodiments, the sample is a biopsy. In some embodiments, the sample isobtained from the subject at a screening session, such as a routineassessment or blood draw to confirm and/or identify the condition ordisease in the subject.

II. CELL THERAPY AND ENGINEERING CELLS

In some embodiments, the cells for use in or administered in connectionwith the provided methods contain or are engineered to contain anengineered receptor, e.g., an engineered antigen receptor, such as achimeric antigen receptor (CAR), or a T cell receptor (TCR). Alsoprovided are populations of such cells, compositions containing suchcells and/or enriched for such cells, such as in which cells of acertain type such as T cells or CD8+ or CD4+ cells are enriched orselected. Among the compositions are pharmaceutical compositions andformulations for administration, such as for adoptive cell therapy. Alsoprovided are therapeutic methods for administering the cells andcompositions to subjects, e.g., patients, in accord with the providedmethods, and/or with the provided articles of manufacture orcompositions.

In some embodiments, the cells include one or more nucleic acidsintroduced via genetic engineering, and thereby express recombinant orgenetically engineered products of such nucleic acids. In someembodiments, gene transfer is accomplished by first stimulating thecells, such as by combining it with a stimulus that induces a responsesuch as proliferation, survival, and/or activation, e.g., as measured byexpression of a cytokine or activation marker, followed by transductionof the activated cells, and expansion in culture to numbers sufficientfor clinical applications.

The cells generally express recombinant receptors, such as antigenreceptors including functional non-TCR antigen receptors, e.g., chimericantigen receptors (CARs), and other antigen-binding receptors such astransgenic T cell receptors (TCRs). Also among the receptors are otherchimeric receptors.

A. Chimeric Antigen Receptors (CARs)

In some embodiments of the provided methods and uses, chimericreceptors, such as a chimeric antigen receptors, contain one or moredomains that combine a ligand-binding domain (e.g. antibody or antibodyfragment) that provides specificity for a desired antigen (e.g., tumorantigen) with intracellular signaling domains. In some embodiments, theintracellular signaling domain is an activating intracellular domainportion, such as a T cell activating domain, providing a primaryactivation signal. In some embodiments, the intracellular signalingdomain contains or additionally contains a costimulatory signalingdomain to facilitate effector functions. In some embodiments, chimericreceptors when genetically engineered into immune cells can modulate Tcell activity, and, in some cases, can modulate T cell differentiationor homeostasis, thereby resulting in genetically engineered cells withimproved longevity, survival and/or persistence in vivo, such as for usein adoptive cell therapy methods.

Exemplary antigen receptors, including CARs, and methods for engineeringand introducing such receptors into cells, include those described, forexample, in international patent application publication numbersWO200014257, WO2013126726, WO2012/129514, WO2014031687, WO2013/166321,WO2013/071154, WO2013/123061 U.S. patent application publication numbersUS2002131960, US2013287748, US20130149337, U.S. Pat. Nos. 6,451,995,7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319,7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118,and European patent application number EP2537416, and/or those describedby Sadelain et al., Cancer Discov. 2013 April; 3(4): 388-398; Davila etal. (2013) PLoS ONE 8(4): e61338; Turtle et al., Curr. Opin. Immunol.,2012 October; 24(5): 633-39; Wu et al., Cancer, 2012 Mar. 18(2): 160-75.In some aspects, the antigen receptors include a CAR as described inU.S. Pat. No. 7,446,190, and those described in International PatentApplication Publication No.: WO/2014055668 A1. Examples of the CARsinclude CARs as disclosed in any of the aforementioned publications,such as WO2014031687, U.S. Pat. Nos. 8,339,645, 7,446,179, US2013/0149337, U.S. Pat. Nos. 7,446,190, 8,389,282, Kochenderfer et al.,2013, Nature Reviews Clinical Oncology, 10, 267-276 (2013); Wang et al.(2012) J. Immunother. 35(9): 689-701; and Brentjens et al., Sci TranslMed. 2013 5(177). See also WO2014031687, U.S. Pat. Nos. 8,339,645,7,446,179, US 2013/0149337, U.S. Pat. Nos. 7,446,190, and 8,389,282.

The chimeric receptors, such as CARs, generally include an extracellularantigen binding domain, such as a portion of an antibody molecule,generally a variable heavy (VH) chain region and/or variable light (VL)chain region of the antibody, e.g., an scFv antibody fragment.

In some embodiments, the antigen targeted by the receptor is apolypeptide. In some embodiments, it is a carbohydrate or othermolecule. In some embodiments, the antigen is selectively expressed oroverexpressed on cells of the disease or condition, e.g., the tumor orpathogenic cells, as compared to normal or non-targeted cells ortissues. In other embodiments, the antigen is expressed on normal cellsand/or is expressed on the engineered cells.

In some embodiments, the antigen is or includes αvP6 integrin (avb6integrin), B cell maturation antigen (BCMA), B7-H3, B7-H6, carbonicanhydrase 9 (CA9, also known as CAIX or G250), a cancer-testis antigen,cancer/testis antigen 1B (CTAG, also known as NY-ESO-1 and LAGE-2),carcinoembryonic antigen (CEA), a cyclin, cyclin A2, C-C Motif ChemokineLigand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44,CD44v6, CD44v7/8, CD123, CD133, CD138, CD171, chondroitin sulfateproteoglycan 4 (CSPG4), epidermal growth factor protein (EGFR), type IIIepidermal growth factor receptor mutation (EGFR vIII), epithelialglycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrinB2,ephrin receptor A2 (EPHa2), estrogen receptor, Fc receptor like 5(FCRL5; also known as Fc receptor homolog 5 or FCRH5), fetalacetylcholine receptor (fetal AchR), a folate binding protein (FBP),folate receptor alpha, ganglioside GD2, O-acetylated GD2 (OGD2),ganglioside GD3, glycoprotein 100 (gp100), glypican-3 (GPC3), G ProteinCoupled Receptor 5D (GPRC5D), Her2/neu (receptor tyrosine kinaseerb-B2), Her3 (erb-B3), Her4 (erb-B4), erbB dimers, Human high molecularweight-melanoma-associated antigen (HMW-MAA), hepatitis B surfaceantigen, Human leukocyte antigen A1 (HLA-A1), Human leukocyte antigen A2(HLA-A2), IL-22 receptor alpha(IL-22Rα), IL-13 receptor alpha 2(IL-13Ra2), kinase insert domain receptor (kdr), kappa light chain, L1cell adhesion molecule (L1-CAM), CE7 epitope of Li-CAM, Leucine RichRepeat Containing 8 Family Member A (LRRC8A), Lewis Y,Melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MAGE-A10,mesothelin (MSLN), c-Met, murine cytomegalovirus (CMV), mucin 1 (MUC1),MUC16, natural killer group 2 member D (NKG2D) ligands, melan A(MART-1), neural cell adhesion molecule (NCAM), oncofetal antigen,Preferentially expressed antigen of melanoma (PRAME), progesteronereceptor, a prostate specific antigen, prostate stem cell antigen(PSCA), prostate specific membrane antigen (PSMA), Receptor TyrosineKinase Like Orphan Receptor 1 (ROR1), survivin, Trophoblast glycoprotein(TPBG also known as 5T4), tumor-associated glycoprotein 72 (TAG72),Tyrosinase related protein 1 (TRP1, also known as TYRP1 or gp75),Tyrosinase related protein 2 (TRP2, also known as dopachrometautomerase, dopachrome delta-isomerase or DCT), vascular endothelialgrowth factor receptor (VEGFR), vascular endothelial growth factorreceptor 2 (VEGFR2), Wilms Tumor 1 (WT-1), a pathogen-specific orpathogen-expressed antigen, or an antigen associated with a universaltag, and/or biotinylated molecules, and/or molecules expressed by HIV,HCV, HBV or other pathogens. Antigens targeted by the receptors in someembodiments include antigens associated with a B cell malignancy, suchas any of a number of known B cell marker. In some embodiments, theantigen is or includes CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33,Igkappa, Iglambda, CD79a, CD79b or CD30.

In some embodiments, the antigen is or includes a pathogen-specific orpathogen-expressed antigen. In some embodiments, the antigen is a viralantigen (such as a viral antigen from HIV, HCV, HBV, etc.), bacterialantigens, and/or parasitic antigens.

Antigens targeted by the receptors in some embodiments include antigensassociated with a B cell malignancy, such as any of a number of known Bcell marker. In some embodiments, the antigen targeted by the receptoris CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, Igkappa, Iglambda,CD79a, CD79b or CD30.

In some embodiments, the antigen or antigen binding domain is CD19. Insome embodiments, the scFv contains a VH and a VL derived from anantibody or an antibody fragment specific to CD19. In some embodiments,the antibody or antibody fragment that binds CD19 is a mouse derivedantibody such as FMC63 and SJ25C1. In some embodiments, the antibody orantibody fragment is a human antibody, e.g., as described in U.S. PatentPublication No. US 2016/0152723.

In some embodiments, the scFv is derived from FMC63. FMC63 generallyrefers to a mouse monoclonal IgG1 antibody raised against Nalm-1 and -16cells expressing CD19 of human origin (Ling, N. R., et al. (1987).Leucocyte typing III. 302). The FMC63 antibody comprises CDRH1 and H2set forth in SEQ ID NOS: 38, 39 respectively, and CDRH3 set forth in SEQID NOS: 40 or 54 and CDRL1 set forth in SEQ ID NOS: 35 and CDR L2 36 or55 and CDR L3 sequences 37 or 34. The FMC63 antibody comprises the heavychain variable region (V_(H)) comprising the amino acid sequence of SEQID NO: 41 and the light chain variable region (V_(L)) comprising theamino acid sequence of SEQ ID NO: 42. In some embodiments, the svFvcomprises a variable light chain containing the CDRL1 sequence of SEQ IDNO:35, a CDRL2 sequence of SEQ ID NO:36, and a CDRL3 sequence of SEQ IDNO:37 and/or a variable heavy chain containing a CDRH1 sequence of SEQID NO:38, a CDRH2 sequence of SEQ ID NO:39, and a CDRH3 sequence of SEQID NO:40. In some embodiments, the scFv comprises a variable heavy chainregion set forth in SEQ ID NO:41 and a variable light chain region setforth in SEQ ID NO:42. In some embodiments, the variable heavy andvariable light chain are connected by a linker. In some embodiments, thelinker is set forth in SEQ ID NO:56. In some embodiments, the scFvcomprises, in order, a V_(H), a linker, and a V_(L). In someembodiments, the scFv comprises, in order, a V_(L), a linker, and aV_(H). In some embodiments, the svFc is encoded by a sequence ofnucleotides set forth in SEQ ID NO:57 or a sequence that exhibits atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% sequence identity to SEQ ID NO:57. In some embodiments, thescFv comprises the sequence of amino acids set forth in SEQ ID NO:43 ora sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ IDNO:43.

In some embodiments the scFv is derived from SJ25C1. SJ25C1 is a mousemonoclonal IgG1 antibody raised against Nalm-1 and -16 cells expressingCD19 of human origin (Ling, N. R., et al. (1987). Leucocyte typing III.302). The SJ25C1 antibody comprises CDRH1, H2 and H3 set forth in SEQ IDNOS: 47-49, respectively, and CDRL1, L2 and L3 sequences set forth inSEQ ID NOS: 44-46, respectively. The SJ25C1 antibody comprises the heavychain variable region (V_(H)) comprising the amino acid sequence of SEQID NO: 50 and the light chain variable region (V_(L)) comprising theamino acid sequence of SEQ ID NO: 51. In some embodiments, the svFvcomprises a variable light chain containing the CDRL1 sequence of SEQ IDNO:44, a CDRL2 sequence of SEQ ID NO: 45, and a CDRL3 sequence of SEQ IDNO:46 and/or a variable heavy chain containing a CDRH1 sequence of SEQID NO:47, a CDRH2 sequence of SEQ ID NO:48, and a CDRH3 sequence of SEQID NO:49. In some embodiments, the scFv comprises a variable heavy chainregion set forth in SEQ ID NO:50 and a variable light chain region setforth in SEQ ID NO:51. In some embodiments, the variable heavy andvariable light chain are connected by a linker. In some embodiments, thelinker is set forth in SEQ ID NO:52. In some embodiments, the scFvcomprises, in order, a V_(H), a linker, and a V_(L). In someembodiments, the scFv comprises, in order, a V_(L), a linker, and aV_(H). In some embodiments, the scFv comprises the sequence of aminoacids set forth in SEQ ID NO:53 or a sequence that exhibits at least85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% sequence identity to SEQ ID NO:53.

In some embodiments, the antigen or antigen binding domain is BCMA. Insome embodiments, the scFv contains a VH and a VL derived from anantibody or an antibody fragment specific to BCMA. In some embodiments,the antibody or antibody fragment that binds BCMA is or contains a VHand a VL from an antibody or antibody fragment set forth inInternational Patent Applications, Publication Number WO 2016/090327 andWO 2016/090320.

In some embodiments, the antigen or antigen binding domain is GPRC5D. Insome embodiments, the scFv contains a VH and a VL derived from anantibody or an antibody fragment specific to GPRC5D. In someembodiments, the antibody or antibody fragment that binds GPRC5D is orcontains a VH and a VL from an antibody or antibody fragment set forthin International Patent Applications, Publication Number WO 2016/090329and WO 2016/090312.

In some embodiments, the antigen is CD20. In some embodiments, the scFvcontains a VH and a VL derived from an antibody or an antibody fragmentspecific to CD20. In some embodiments, the antibody or antibody fragmentthat binds CD20 is an antibody that is or is derived from Rituximab,such as is Rituximab scFv.

In some embodiments, the antigen is CD22. In some embodiments, the scFvcontains a VH and a VL derived from an antibody or an antibody fragmentspecific to CD22. In some embodiments, the antibody or antibody fragmentthat binds CD22 is an antibody that is or is derived from m971, such asis m971 scFv.

In some embodiments, the antigen or antigen binding domain is BCMA. Insome embodiments, the scFv contains a VH and a VL derived from anantibody or an antibody fragment specific to BCMA. In some embodiments,the antibody or antibody fragment that binds BCMA is or contains a VHand a VL from an antibody or antibody fragment set forth inInternational Patent Applications, Publication Number WO 2016/090327 andWO 2016/090320.

In some embodiments, the antigen or antigen binding domain is GPRC5D. Insome embodiments, the scFv contains a VH and a VL derived from anantibody or an antibody fragment specific to GPRC5D. In someembodiments, the antibody or antibody fragment that binds GPRC5D is orcontains a VH and a VL from an antibody or antibody fragment set forthin International Patent Applications, Publication Number WO 2016/090329and WO 2016/090312.

In some embodiments, the chimeric antigen receptor includes anextracellular portion containing an antibody or antibody fragment. Insome aspects, the chimeric antigen receptor includes an extracellularportion containing the antibody or fragment and an intracellularsignaling domain. In some embodiments, the antibody or fragment includesan scFv.

In some embodiments, the antibody portion of the recombinant receptor,e.g., CAR, further includes at least a portion of an immunoglobulinconstant region, such as a hinge region, e.g., an IgG4 hinge region,and/or a CH1/CL and/or Fc region. In some embodiments, the constantregion or portion is of a human IgG, such as IgG4 or IgG1. In someaspects, the portion of the constant region serves as a spacer regionbetween the antigen-recognition component, e.g., scFv, and transmembranedomain. The spacer can be of a length that provides for increasedresponsiveness of the cell following antigen binding, as compared to inthe absence of the spacer. Exemplary spacers include, but are notlimited to, those described in Hudecek et al. (2013) Clin. Cancer Res.,19:3153, international patent application publication numberWO2014031687, U.S. Pat. No. 8,822,647 or published app. No.US2014/0271635.

In some embodiments, the constant region or portion is of a human IgG,such as IgG4 or IgG1. In some embodiments, the spacer has the sequenceESKYGPPCPPCP (set forth in SEQ ID NO: 1), and is encoded by the sequenceset forth in SEQ ID NO: 2. In some embodiments, the spacer has thesequence set forth in SEQ ID NO: 3. In some embodiments, the spacer hasthe sequence set forth in SEQ ID NO: 4. In some embodiments, theconstant region or portion is of IgD. In some embodiments, the spacerhas the sequence set forth in SEQ ID NO: 5. In some embodiments, thespacer has a sequence of amino acids that exhibits at least 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity to any of SEQ ID NOS: 1, 3, 4 or 5. In someembodiments, the spacer has the sequence set forth in SEQ ID NOS: 27-33.In some embodiments, the spacer has a sequence of amino acids thatexhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOS:27-33, 58, 59.

In some embodiments, the antigen receptor comprises an intracellulardomain linked directly or indirectly to the extracellular domain. Insome embodiments, the chimeric antigen receptor includes a transmembranedomain linking the extracellular domain and the intracellular signalingdomain. In some embodiments, the intracellular signaling domaincomprises an ITAM. For example, in some aspects, the antigen recognitiondomain (e.g. extracellular domain) generally is linked to one or moreintracellular signaling components, such as signaling components thatmimic activation through an antigen receptor complex, such as a TCRcomplex, in the case of a CAR, and/or signal via another cell surfacereceptor. In some embodiments, the chimeric receptor comprises atransmembrane domain linked or fused between the extracellular domain(e.g. scFv) and intracellular signaling domain. Thus, in someembodiments, the antigen-binding component (e.g., antibody) is linked toone or more transmembrane and intracellular signaling domains.

In one embodiment, a transmembrane domain that naturally is associatedwith one of the domains in the receptor, e.g., CAR, is used. In someinstances, the transmembrane domain is selected or modified by aminoacid substitution to avoid binding of such domains to the transmembranedomains of the same or different surface membrane proteins to minimizeinteractions with other members of the receptor complex.

The transmembrane domain in some embodiments is derived either from anatural or from a synthetic source. Where the source is natural, thedomain in some aspects is derived from any membrane-bound ortransmembrane protein. Transmembrane regions include those derived from(i.e. comprise at least the transmembrane region(s) of) the alpha, betaor zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5,CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.Alternatively the transmembrane domain in some embodiments is synthetic.In some aspects, the synthetic transmembrane domain comprisespredominantly hydrophobic residues such as leucine and valine. In someaspects, a triplet of phenylalanine, tryptophan and valine will be foundat each end of a synthetic transmembrane domain. In some embodiments,the linkage is by linkers, spacers, and/or transmembrane domain(s). Insome aspects, the transmembrane domain contains a transmembrane portionof CD28.

In some embodiments, the extracellular domain and transmembrane domaincan be linked directly or indirectly. In some embodiments, theextracellular domain and transmembrane are linked by a spacer, such asany described herein. In some embodiments, the receptor containsextracellular portion of the molecule from which the transmembranedomain is derived, such as a CD28 extracellular portion.

Among the intracellular signaling domains are those that mimic orapproximate a signal through a natural antigen receptor, a signalthrough such a receptor in combination with a costimulatory receptor,and/or a signal through a costimulatory receptor alone. In someembodiments, a short oligo- or polypeptide linker, for example, a linkerof between 2 and 10 amino acids in length, such as one containingglycines and serines, e.g., glycine-serine doublet, is present and formsa linkage between the transmembrane domain and the cytoplasmic signalingdomain of the CAR.

T cell activation is in some aspects described as being mediated by twoclasses of cytoplasmic signaling sequences: those that initiateantigen-dependent primary activation through the TCR (primarycytoplasmic signaling sequences), and those that act in anantigen-independent manner to provide a secondary or co-stimulatorysignal (secondary cytoplasmic signaling sequences). In some aspects, theCAR includes one or both of such signaling components.

The receptor, e.g., the CAR, generally includes at least oneintracellular signaling component or components. In some aspects, theCAR includes a primary cytoplasmic signaling sequence that regulatesprimary activation of the TCR complex. Primary cytoplasmic signalingsequences that act in a stimulatory manner may contain signaling motifswhich are known as immunoreceptor tyrosine-based activation motifs orITAMs. Examples of ITAM containing primary cytoplasmic signalingsequences include those derived from CD3 zeta chain, FcR gamma, CD3gamma, CD3 delta and CD3 epsilon. In some embodiments, cytoplasmicsignaling molecule(s) in the CAR contain(s) a cytoplasmic signalingdomain, portion thereof, or sequence derived from CD3 zeta.

In some embodiments, the receptor includes an intracellular component ofa TCR complex, such as a TCR CD3 chain that mediates T-cell activationand cytotoxicity, e.g., CD3 zeta chain. Thus, in some aspects, theantigen-binding portion is linked to one or more cell signaling modules.In some embodiments, cell signaling modules include CD3 transmembranedomain, CD3 intracellular signaling domains, and/or other CD3transmembrane domains. In some embodiments, the receptor, e.g., CAR,further includes a portion of one or more additional molecules such asFc receptor γ, CD8, CD4, CD25, or CD16. For example, in some aspects,the CAR or other chimeric receptor includes a chimeric molecule betweenCD3-zeta (CD3-ζ) or Fc receptor γ and CD8, CD4, CD25 or CD16.

In some embodiments, upon ligation of the CAR or other chimericreceptor, the cytoplasmic domain or intracellular signaling domain ofthe receptor activates at least one of the normal effector functions orresponses of the immune cell, e.g., T cell engineered to express theCAR. For example, in some contexts, the CAR induces a function of a Tcell such as cytolytic activity or T-helper activity, such as secretionof cytokines or other factors. In some embodiments, a truncated portionof an intracellular signaling domain of an antigen receptor component orcostimulatory molecule is used in place of an intact immunostimulatorychain, for example, if it transduces the effector function signal. Insome embodiments, the intracellular signaling domain or domains includethe cytoplasmic sequences of the T cell receptor (TCR), and in someaspects also those of co-receptors that in the natural context act inconcert with such receptors to initiate signal transduction followingantigen receptor engagement.

In the context of a natural TCR, full activation generally requires notonly signaling through the TCR, but also a costimulatory signal. Thus,in some embodiments, to promote full activation, a component forgenerating secondary or co-stimulatory signal is also included in theCAR. In other embodiments, the CAR does not include a component forgenerating a costimulatory signal. In some aspects, an additional CAR isexpressed in the same cell and provides the component for generating thesecondary or costimulatory signal.

In some embodiments, the chimeric antigen receptor contains anintracellular domain of a T cell costimulatory molecule. In someembodiments, the CAR includes a signaling domain and/or transmembraneportion of a costimulatory receptor, such as CD28, 4-1BB, OX40, DAP10,and ICOS. In some aspects, the same CAR includes both the activating andcostimulatory components. In some embodiments, the chimeric antigenreceptor contains an intracellular domain derived from a T cellcostimulatory molecule or a functional variant thereof, such as betweenthe transmembrane domain and intracellular signaling domain. In someaspects, the T cell costimulatory molecule is CD28 or 41BB.

In some embodiments, the activating domain is included within one CAR,whereas the costimulatory component is provided by another CARrecognizing another antigen. In some embodiments, the CARs includeactivating or stimulatory CARs, costimulatory CARs, both expressed onthe same cell (see WO2014/055668). In some aspects, the cells includeone or more stimulatory or activating CAR and/or a costimulatory CAR. Insome embodiments, the cells further include inhibitory CARs (iCARs, seeFedorov et al., Sci. Transl. Medicine, 5(215) (December, 2013), such asa CAR recognizing an antigen other than the one associated with and/orspecific for the disease or condition whereby an activating signaldelivered through the disease-targeting CAR is diminished or inhibitedby binding of the inhibitory CAR to its ligand, e.g., to reduceoff-target effects.

In some embodiments, the two receptors induce, respectively, anactivating and an inhibitory signal to the cell, such that ligation ofone of the receptor to its antigen activates the cell or induces aresponse, but ligation of the second inhibitory receptor to its antigeninduces a signal that suppresses or dampens that response. Examples arecombinations of activating CARs and inhibitory CARs (iCARs). Such astrategy may be used, for example, to reduce the likelihood ofoff-target effects in the context in which the activating CAR binds anantigen expressed in a disease or condition but which is also expressedon normal cells, and the inhibitory receptor binds to a separate antigenwhich is expressed on the normal cells but not cells of the disease orcondition.

In some aspects, the chimeric receptor is or includes an inhibitory CAR(e.g. iCAR) and includes intracellular components that dampen orsuppress an immune response, such as an ITAM- and/or costimulatory-promoted response in the cell. Exemplary of suchintracellular signaling components are those found on immune checkpointmolecules, including PD-1, CTLA4, LAG3, BTLA, OX2R, TIM-3, TIGIT,LAIR-1, PGE2 receptors, EP2/4 Adenosine receptors including A2AR. Insome aspects, the engineered cell includes an inhibitory CAR including asignaling domain of or derived from such an inhibitory molecule, suchthat it serves to dampen the response of the cell, for example, thatinduced by an activating and/or costimulatory CAR.

In certain embodiments, the intracellular signaling domain comprises aCD28 transmembrane and signaling domain linked to a CD3 (e.g., CD3-zeta)intracellular domain. In some embodiments, the intracellular signalingdomain comprises a chimeric CD28 and CD137 (4-1BB, TNFRSF9)co-stimulatory domains, linked to a CD3 zeta intracellular domain.

In some embodiments, the CAR encompasses one or more, e.g., two or more,costimulatory domains and an activation domain, e.g., primary activationdomain, in the cytoplasmic portion. Exemplary CARs include intracellularcomponents of CD3-zeta, CD28, and 4-1BB.

In some embodiments, the antigen receptor further includes a markerand/or cells expressing the CAR or other antigen receptor furtherincludes a surrogate marker, such as a cell surface marker, which may beused to confirm transduction or engineering of the cell to express thereceptor. In some aspects, the marker includes all or part (e.g.,truncated form) of CD34, a NGFR, or epidermal growth factor receptor,such as truncated version of such a cell surface receptor (e.g., tEGFR).In some embodiments, the nucleic acid encoding the marker is operablylinked to a polynucleotide encoding for a linker sequence, such as acleavable linker sequence, e.g., T2A. For example, a marker, andoptionally a linker sequence, can be any as disclosed in publishedpatent application No. WO2014031687. For example, the marker can be atruncated EGFR (tEGFR) that is, optionally, linked to a linker sequence,such as a T2A cleavable linker sequence.

An exemplary polypeptide for a truncated EGFR (e.g. tEGFR) comprises thesequence of amino acids set forth in SEQ ID NO: 7 or 16 or a sequence ofamino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQID NO: 7 or 16. An exemplary T2A linker sequence comprises the sequenceof amino acids set forth in SEQ ID NO: 6 or 17 or a sequence of aminoacids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ IDNO: 6 or 17.

In some embodiments, the marker is a molecule, e.g., cell surfaceprotein, not naturally found on T cells or not naturally found on thesurface of T cells, or a portion thereof. In some embodiments, themolecule is a non-self molecule, e.g., non-self protein, i.e., one thatis not recognized as “self” by the immune system of the host into whichthe cells will be adoptively transferred.

In some embodiments, the marker serves no therapeutic function and/orproduces no effect other than to be used as a marker for geneticengineering, e.g., for selecting cells successfully engineered. In otherembodiments, the marker may be a therapeutic molecule or moleculeotherwise exerting some desired effect, such as a ligand for a cell tobe encountered in vivo, such as a costimulatory or immune checkpointmolecule to enhance and/or dampen responses of the cells upon adoptivetransfer and encounter with ligand.

In some cases, CARs are referred to as first, second, and/or thirdgeneration CARs. In some aspects, a first generation CAR is one thatsolely provides a CD3-chain induced signal upon antigen binding; in someaspects, a second-generation CARs is one that provides such a signal andcostimulatory signal, such as one including an intracellular signalingdomain from a costimulatory receptor such as CD28 or CD137; in someaspects, a third generation CAR is one that includes multiplecostimulatory domains of different costimulatory receptors.

For example, in some embodiments, the CAR contains an antibody, e.g., anantibody fragment, such as an scFv, specific to an antigen including anyas described, a transmembrane domain that is or contains a transmembraneportion of CD28 or a functional variant thereof, and an intracellularsignaling domain containing a signaling portion of CD28 or functionalvariant thereof and a signaling portion of CD3 zeta or functionalvariant thereof. In some embodiments, the CAR contains an antibody,e.g., antibody fragment, such as an scFv, specific to an antigenincluding any as described, a transmembrane domain that is or contains atransmembrane portion of CD28 or a functional variant thereof, and anintracellular signaling domain containing a signaling portion of a 4-1BBor functional variant thereof and a signaling portion of CD3 zeta orfunctional variant thereof. In some such embodiments, the receptorfurther includes a spacer containing a portion of an Ig molecule, suchas a human Ig molecule, such as an Ig hinge, e.g. an IgG4 hinge, such asa hinge-only spacer.

In some embodiments, the transmembrane domain of the recombinantreceptor, e.g., the CAR, is or includes a transmembrane domain of humanCD28 (e.g. Accession No. P01747.1) or variant thereof, such as atransmembrane domain that comprises the sequence of amino acids setforth in SEQ ID NO: 8 or a sequence of amino acids that exhibits atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or more sequence identity to SEQ ID NO: 8; in some embodiments,the transmembrane-domain containing portion of the recombinant receptorcomprises the sequence of amino acids set forth in SEQ ID NO: 9 or asequence of amino acids having at least at or about 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity thereto.

In some embodiments, the intracellular signaling component(s) of therecombinant receptor, e.g. the CAR, contains an intracellularcostimulatory signaling domain of human CD28 or a functional variant orportion thereof, such as a domain with an LL to GG substitution atpositions 186-187 of a native CD28 protein. For example, theintracellular signaling domain can comprise the sequence of amino acidsset forth in SEQ ID NO: 10 or 11 or a sequence of amino acids thatexhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 10 or 11. Insome embodiments, the intracellular domain comprises an intracellularcostimulatory signaling domain of 4-1BB (e.g. (Accession No. Q07011.1)or functional variant or portion thereof, such as the sequence of aminoacids set forth in SEQ ID NO: 12 or a sequence of amino acids thatexhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 12.

In some embodiments, the intracellular signaling domain of therecombinant receptor, e.g. the CAR, comprises a human CD3 zetastimulatory signaling domain or functional variant thereof, such as an112 AA cytoplasmic domain of isoform 3 of human CD3ζ (Accession No.:P20963.2) or a CD3 zeta signaling domain as described in U.S. Pat. No.7,446,190 or 8,911,993. For example, in some embodiments, theintracellular signaling domain comprises the sequence of amino acids asset forth in SEQ ID NO: 13, 14 or 15 or a sequence of amino acids thatexhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 13, 14 or 15.

In some aspects, the spacer contains only a hinge region of an IgG, suchas only a hinge of IgG4 or IgG1, such as the hinge only spacer set forthin SEQ ID NO: 1. In other embodiments, the spacer is or contains an Ighinge, e.g., an IgG4-derived hinge, optionally linked to a CH2 and/orCH3 domains. In some embodiments, the spacer is an Ig hinge, e.g., anIgG4 hinge, linked to CH2 and CH3 domains, such as set forth in SEQ IDNO: 4. In some embodiments, the spacer is an Ig hinge, e.g., an IgG4hinge, linked to a CH3 domain only, such as set forth in SEQ ID NO: 3.In some embodiments, the spacer is or comprises a glycine-serine richsequence or other flexible linker such as known flexible linkers.

For example, in some embodiments, the CAR includes an antibody such asan antibody fragment, including scFvs, a spacer, such as a spacercontaining a portion of an immunoglobulin molecule, such as a hingeregion and/or one or more constant regions of a heavy chain molecule,such as an Ig-hinge containing spacer, a transmembrane domain containingall or a portion of a CD28-derived transmembrane domain, a CD28-derivedintracellular signaling domain, and a CD3 zeta signaling domain. In someembodiments, the CAR includes an antibody or fragment, such as scFv, aspacer such as any of the Ig-hinge containing spacers, a CD28-derivedtransmembrane domain, a 4-1BB-derived intracellular signaling domain,and a CD3 zeta-derived signaling domain.

In some embodiments, nucleic acid molecules encoding such CAR constructsfurther includes a sequence encoding a T2A ribosomal skip element and/ora surrogate marker (e.g., tEGFR sequence), e.g., downstream of thesequence encoding the CAR. In some embodiments, the sequence encodes aT2A ribosomal skip element set forth in SEQ ID NO: 6 or 17, or asequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity to SEQ ID NO: 6 or 17.

Exemplary surrogate markers can include truncated forms of cell surfacepolypeptides, such as truncated forms that are non-functional and to nottransduce or are not capable of transducing a signal or a signalordinarily transduced by the full-length form of the cell surfacepolypeptide, and/or do not or are not capable of internalizing.Exemplary truncated cell surface polypeptides including truncated formsof growth factors or other receptors such as a truncated human epidermalgrowth factor receptor 2 (tHER2), a truncated epidermal growth factorreceptor (tEGFR, exemplary tEGFR sequence set forth in 7 or 16) or aprostate-specific membrane antigen (PSMA) or modified form thereof.tEGFR may contain an epitope recognized by the antibody cetuximab(Erbitux®) or other therapeutic anti-EGFR antibody or binding molecule,which can be used to identify or select cells that have been engineeredto express the tEGFR construct and an encoded exogenous protein, and/orto eliminate or separate cells expressing the encoded exogenous protein.See U.S. Pat. No. 8,802,374 and Liu et al., Nature Biotech. 2016 April;34(4): 430-434). In some aspects, the marker, e.g. surrogate marker,includes all or part (e.g., truncated form) of CD34, a NGFR, a CD19 or atruncated CD19, e.g., a truncated non-human CD19, or epidermal growthfactor receptor (e.g., tEGFR). In some embodiments, the marker is orcomprises a fluorescent protein, such as green fluorescent protein(GFP), enhanced green fluorescent protein (EGFP), such as super-fold GFP(sfGFP), red fluorescent protein (RFP), such as tdTomato, mCherry,mStrawberry, AsRed2, DsRed or DsRed2, cyan fluorescent protein (CFP),blue green fluorescent protein (BFP), enhanced blue fluorescent protein(EBFP), and yellow fluorescent protein (YFP), and variants thereof,including species variants, monomeric variants, and codon-optimizedand/or enhanced variants of the fluorescent proteins. In someembodiments, the marker is or comprises an enzyme, such as a luciferase,the lacZ gene from E. coli, alkaline phosphatase, secreted embryonicalkaline phosphatase (SEAP), chloramphenicol acetyl transferase (CAT).Exemplary light-emitting reporter genes include luciferase (luc),β-galactosidase, chloramphenicol acetyltransferase (CAT),β-glucuronidase (GUS) or variants thereof.

In some embodiments, the marker is a selection marker. In someembodiments, the selection marker is or comprises a polypeptide thatconfers resistance to exogenous agents or drugs. In some embodiments,the selection marker is an antibiotic resistance gene. In someembodiments, the selection marker is an antibiotic resistance geneconfers antibiotic resistance to a mammalian cell. In some embodiments,the selection marker is or comprises a Puromycin resistance gene, aHygromycin resistance gene, a Blasticidin resistance gene, a Neomycinresistance gene, a Geneticin resistance gene or a Zeocin resistance geneor a modified form thereof.

In some embodiments, the nucleic acid encoding the marker is operablylinked to a polynucleotide encoding for a linker sequence, such as acleavable linker sequence, e.g., a T2A. For example, a marker, andoptionally a linker sequence, can be any as disclosed in PCT Pub. No.WO2014031687.

In some embodiments, T cells expressing an antigen receptor (e.g. CAR)can also be generated to express a truncated EGFR (EGFRt) as anon-immunogenic selection epitope (e.g. by introduction of a constructencoding the CAR and EGFRt separated by a T2A ribosome switch to expresstwo proteins from the same construct), which then can be used as amarker to detect such cells (see e.g. U.S. Pat. No. 8,802,374). In someembodiments, the sequence encodes an tEGFR sequence set forth in SEQ IDNO: 7 or 16, or a sequence of amino acids that exhibits at least 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity to SEQ ID NO: 7 or 16. In some cases, thepeptide, such as T2A, can cause the ribosome to skip (ribosome skipping)synthesis of a peptide bond at the C-terminus of a 2A element, leadingto separation between the end of the 2A sequence and the next peptidedownstream (see, for example, de Felipe. Genetic Vaccines and Ther. 2:13(2004) and deFelipe et al. Traffic 5:616-626 (2004)). Many 2A elementsare known. Examples of 2A sequences that can be used in the methods andnucleic acids disclosed herein, without limitation, 2A sequences fromthe foot-and-mouth disease virus (F2A, e.g., SEQ ID NO: 21), equinerhinitis A virus (E2A, e.g., SEQ ID NO: 20), Thosea asigna virus (T2A,e.g., SEQ ID NO: 6 or 17), and porcine teschovirus-1 (P2A, e.g., SEQ IDNO: 18 or 19) as described in U.S. Patent Publication No. 20070116690.

The recombinant receptors, such as CARs, expressed by the cellsadministered to the subject generally recognize or specifically bind toa molecule that is expressed in, associated with, and/or specific forthe disease or condition or cells thereof being treated. Upon specificbinding to the molecule, e.g., antigen, the receptor generally deliversan immunostimulatory signal, such as an ITAM-transduced signal, into thecell, thereby promoting an immune response targeted to the disease orcondition. For example, in some embodiments, the cells express a CARthat specifically binds to an antigen expressed by a cell or tissue ofthe disease or condition or associated with the disease or condition.

B. TCRs

In some embodiments, engineered cells, such as T cells, are providedthat express a T cell receptor (TCR) or antigen-binding portion thereofthat recognizes an peptide epitope or T cell epitope of a targetpolypeptide, such as an antigen of a tumor, viral or autoimmune protein.

In some embodiments, a “T cell receptor” or “TCR” is a molecule thatcontains a variable α and β chains (also known as TCRα and TCRβ,respectively) or a variable γ and δ chains (also known as TCRα and TCRβ,respectively), or antigen-binding portions thereof, and which is capableof specifically binding to a peptide bound to an MHC molecule. In someembodiments, the TCR is in the a form. Typically, TCRs that exist in αβand γδ forms are generally structurally similar, but T cells expressingthem may have distinct anatomical locations or functions. A TCR can befound on the surface of a cell or in soluble form. Generally, a TCR isfound on the surface of T cells (or T lymphocytes) where it is generallyresponsible for recognizing antigens bound to major histocompatibilitycomplex (MHC) molecules.

Unless otherwise stated, the term “TCR” should be understood toencompass full TCRs as well as antigen-binding portions orantigen-binding fragments thereof. In some embodiments, the TCR is anintact or full-length TCR, including TCRs in the a form or 76 form. Insome embodiments, the TCR is an antigen-binding portion that is lessthan a full-length TCR but that binds to a specific peptide bound in anMHC molecule, such as binds to an MHC-peptide complex. In some cases, anantigen-binding portion or fragment of a TCR can contain only a portionof the structural domains of a full-length or intact TCR, but yet isable to bind the peptide epitope, such as MHC-peptide complex, to whichthe full TCR binds. In some cases, an antigen-binding portion containsthe variable domains of a TCR, such as variable a chain and variable βchain of a TCR, sufficient to form a binding site for binding to aspecific MHC-peptide complex. Generally, the variable chains of a TCRcontain complementarity determining regions involved in recognition ofthe peptide, MHC and/or MHC-peptide complex.

In some embodiments, the variable domains of the TCR containhypervariable loops, or complementarity determining regions (CDRs),which generally are the primary contributors to antigen recognition andbinding capabilities and specificity. In some embodiments, a CDR of aTCR or combination thereof forms all or substantially all of theantigen-binding site of a given TCR molecule. The various CDRs within avariable region of a TCR chain generally are separated by frameworkregions (FRs), which generally display less variability among TCRmolecules as compared to the CDRs (see, e.g., Jores et al., Proc. Nat'lAcad. Sci. U.S.A. 87:9138, 1990; Chothia et al., EMBO J. 7:3745, 1988;see also Lefranc et al., Dev. Comp. Immunol. 27:55, 2003). In someembodiments, CDR3 is the main CDR responsible for antigen binding orspecificity, or is the most important among the three CDRs on a givenTCR variable region for antigen recognition, and/or for interaction withthe processed peptide portion of the peptide-MHC complex. In somecontexts, the CDR1 of the alpha chain can interact with the N-terminalpart of certain antigenic peptides. In some contexts, CDR1 of the betachain can interact with the C-terminal part of the peptide. In somecontexts, CDR2 contributes most strongly to or is the primary CDRresponsible for the interaction with or recognition of the MHC portionof the MHC-peptide complex. In some embodiments, the variable region ofthe β-chain can contain a further hypervariable region (CDR4 or HVR4),which generally is involved in superantigen binding and not antigenrecognition (Kotb (1995) Clinical Microbiology Reviews, 8:411-426).

In some embodiments, a TCR also can contain a constant domain, atransmembrane domain and/or a short cytoplasmic tail (see, e.g., Janewayet al., Immunobiology: The Immune System in Health and Disease, 3rd Ed.,Current Biology Publications, p. 4:33, 1997). In some aspects, eachchain of the TCR can possess one N-terminal immunoglobulin variabledomain, one immunoglobulin constant domain, a transmembrane region, anda short cytoplasmic tail at the C-terminal end. In some embodiments, aTCR is associated with invariant proteins of the CD3 complex involved inmediating signal transduction.

In some embodiments, a TCR chain contains one or more constant domain.For example, the extracellular portion of a given TCR chain (e.g.,α-chain or β-chain) can contain two immunoglobulin-like domains, such asa variable domain (e.g., Vα or Vβ; typically amino acids 1 to 116 basedon Kabat numbering Kabat et al., “Sequences of Proteins of ImmunologicalInterest, US Dept. Health and Human Services, Public Health ServiceNational Institutes of Health, 1991, 5th ed.) and a constant domain(e.g., a-chain constant domain or Cα, typically positions 117 to 259 ofthe chain based on Kabat numbering or chain constant domain or C_(β),typically positions 117 to 295 of the chain based on Kabat) adjacent tothe cell membrane. For example, in some cases, the extracellular portionof the TCR formed by the two chains contains two membrane-proximalconstant domains, and two membrane-distal variable domains, whichvariable domains each contain CDRs. The constant domain of the TCR maycontain short connecting sequences in which a cysteine residue forms adisulfide bond, thereby linking the two chains of the TCR. In someembodiments, a TCR may have an additional cysteine residue in each ofthe α and β chains, such that the TCR contains two disulfide bonds inthe constant domains.

In some embodiments, the TCR chains contain a transmembrane domain. Insome embodiments, the transmembrane domain is positively charged. Insome cases, the TCR chain contains a cytoplasmic tail. In some cases,the structure allows the TCR to associate with other molecules like CD3and subunits thereof. For example, a TCR containing constant domainswith a transmembrane region may anchor the protein in the cell membraneand associate with invariant subunits of the CD3 signaling apparatus orcomplex. The intracellular tails of CD3 signaling subunits (e.g. CD3γ,CD3δ, CD3ε and CD3ζ chains) contain one or more immunoreceptortyrosine-based activation motif or ITAM that are involved in thesignaling capacity of the TCR complex.

In some embodiments, the TCR may be a heterodimer of two chains α and β(or optionally γ and δ) or it may be a single chain TCR construct. Insome embodiments, the TCR is a heterodimer containing two separatechains (α and β chains or γ and δ chains) that are linked, such as by adisulfide bond or disulfide bonds.

In some embodiments, the TCR can be generated from a known TCRsequence(s), such as sequences of Vα,β chains, for which a substantiallyfull-length coding sequence is readily available. Methods for obtainingfull-length TCR sequences, including V chain sequences, from cellsources are well known. In some embodiments, nucleic acids encoding theTCR can be obtained from a variety of sources, such as by polymerasechain reaction (PCR) amplification of TCR-encoding nucleic acids withinor isolated from a given cell or cells, or synthesis of publiclyavailable TCR DNA sequences.

In some embodiments, the TCR is obtained from a biological source, suchas from cells such as from a T cell (e.g. cytotoxic T cell), T-cellhybridomas or other publicly available source. In some embodiments, theT-cells can be obtained from in vivo isolated cells. In someembodiments, the TCR is a thymically selected TCR. In some embodiments,the TCR is a neoepitope-restricted TCR. In some embodiments, the T-cellscan be a cultured T-cell hybridoma or clone. In some embodiments, theTCR or antigen-binding portion thereof or antigen-binding fragmentthereof can be synthetically generated from knowledge of the sequence ofthe TCR.

In some embodiments, the TCR is generated from a TCR identified orselected from screening a library of candidate TCRs against a targetpolypeptide antigen, or target T cell epitope thereof. TCR libraries canbe generated by amplification of the repertoire of Va and V from T cellsisolated from a subject, including cells present in PBMCs, spleen orother lymphoid organ. In some cases, T cells can be amplified fromtumor-infiltrating lymphocytes (TILs). In some embodiments, TCRlibraries can be generated from CD4+ or CD8+ cells. In some embodiments,the TCRs can be amplified from a T cell source of a normal of healthysubject, i.e. normal TCR libraries. In some embodiments, the TCRs can beamplified from a T cell source of a diseased subject, i.e. diseased TCRlibraries. In some embodiments, degenerate primers are used to amplifythe gene repertoire of Vα and Vβ, such as by RT-PCR in samples, such asT cells, obtained from humans. In some embodiments, scTv libraries canbe assembled from naïve Vα and Vβ libraries in which the amplifiedproducts are cloned or assembled to be separated by a linker. Dependingon the source of the subject and cells, the libraries can be HLAallele-specific. Alternatively, in some embodiments, TCR libraries canbe generated by mutagenesis or diversification of a parent or scaffoldTCR molecule. In some aspects, the TCRs are subjected to directedevolution, such as by mutagenesis, e.g., of the α or β chain. In someaspects, particular residues within CDRs of the TCR are altered. In someembodiments, selected TCRs can be modified by affinity maturation. Insome embodiments, antigen-specific T cells may be selected, such as byscreening to assess CTL activity against the peptide. In some aspects,TCRs, e.g. present on the antigen-specific T cells, may be selected,such as by binding activity, e.g., particular affinity or avidity forthe antigen.

In some embodiments, the TCR or antigen-binding portion thereof is onethat has been modified or engineered. In some embodiments, directedevolution methods are used to generate TCRs with altered properties,such as with higher affinity for a specific MHC-peptide complex. In someembodiments, directed evolution is achieved by display methodsincluding, but not limited to, yeast display (Holler et al. (2003) NatImmunol, 4, 55-62; Holler et al. (2000) Proc Natl Acad Sci USA, 97,5387-92), phage display (Li et al. (2005) Nat Biotechnol, 23, 349-54),or T cell display (Chervin et al. (2008) J Immunol Methods, 339,175-84). In some embodiments, display approaches involve engineering, ormodifying, a known, parent or reference TCR. For example, in some cases,a wild-type TCR can be used as a template for producing mutagenized TCRsin which in one or more residues of the CDRs are mutated, and mutantswith an desired altered property, such as higher affinity for a desiredtarget antigen, are selected.

In some embodiments, peptides of a target polypeptide for use inproducing or generating a TCR of interest are known or can be readilyidentified. In some embodiments, peptides suitable for use in generatingTCRs or antigen-binding portions can be determined based on the presenceof an HLA-restricted motif in a target polypeptide of interest, such asa target polypeptide described below. In some embodiments, peptides areidentified using available computer prediction models. In someembodiments, for predicting MHC class I binding sites, such modelsinclude, but are not limited to, ProPred1 (Singh and Raghava (2001)Bioinformatics 17(12):1236-1237, and SYFPEITHI (see Schuler et al.(2007) Immunoinformatics Methods in Molecular Biology, 409(1): 75-932007). In some embodiments, the MHC-restricted epitope is HLA-A0201,which is expressed in approximately 39-46% of all Caucasians andtherefore, represents a suitable choice of MHC antigen for use preparinga TCR or other MHC-peptide binding molecule.

HLA-A0201-binding motifs and the cleavage sites for proteasomes andimmune-proteasomes using computer prediction models are known. Forpredicting MHC class I binding sites, such models include, but are notlimited to, ProPred1 (described in more detail in Singh and Raghava,ProPred: prediction of HLA-DR binding sites. BIOINFORMATICS17(12):1236-1237 2001), and SYFPEITHI (see Schuler et al. SYFPEITHI,Database for Searching and T-Cell Epitope Prediction. inImmunoinformatics Methods in Molecular Biology, vol 409(1): 75-93 2007)

In some embodiments, the TCR or antigen binding portion thereof may be arecombinantly produced natural protein or mutated form thereof in whichone or more property, such as binding characteristic, has been altered.In some embodiments, a TCR may be derived from one of various animalspecies, such as human, mouse, rat, or other mammal. A TCR may becell-bound or in soluble form. In some embodiments, for purposes of theprovided methods, the TCR is in cell-bound form expressed on the surfaceof a cell.

In some embodiments, the TCR is a full-length TCR. In some embodiments,the TCR is an antigen-binding portion. In some embodiments, the TCR is adimeric TCR (dTCR). In some embodiments, the TCR is a single-chain TCR(sc-TCR). In some embodiments, a dTCR or scTCR have the structures asdescribed in WO 03/020763, WO 04/033685, WO2011/044186.

In some embodiments, the TCR contains a sequence corresponding to thetransmembrane sequence. In some embodiments, the TCR does contain asequence corresponding to cytoplasmic sequences. In some embodiments,the TCR is capable of forming a TCR complex with CD3. In someembodiments, any of the TCRs, including a dTCR or scTCR, can be linkedto signaling domains that yield an active TCR on the surface of a Tcell. In some embodiments, the TCR is expressed on the surface of cells.

In some embodiments a dTCR contains a first polypeptide wherein asequence corresponding to a TCR α chain variable region sequence isfused to the N terminus of a sequence corresponding to a TCR α chainconstant region extracellular sequence, and a second polypeptide whereina sequence corresponding to a TCR β chain variable region sequence isfused to the N terminus a sequence corresponding to a TCR β chainconstant region extracellular sequence, the first and secondpolypeptides being linked by a disulfide bond. In some embodiments, thebond can correspond to the native inter-chain disulfide bond present innative dimeric a TCRs. In some embodiments, the interchain disulfidebonds are not present in a native TCR. For example, in some embodiments,one or more cysteines can be incorporated into the constant regionextracellular sequences of dTCR polypeptide pair. In some cases, both anative and a non-native disulfide bond may be desirable. In someembodiments, the TCR contains a transmembrane sequence to anchor to themembrane.

In some embodiments, a dTCR contains a TCR α chain containing a variablea domain, a constant α domain and a first dimerization motif attached tothe C-terminus of the constant α domain, and a TCR β chain comprising avariable β domain, a constant β domain and a first dimerization motifattached to the C-terminus of the constant β domain, wherein the firstand second dimerization motifs easily interact to form a covalent bondbetween an amino acid in the first dimerization motif and an amino acidin the second dimerization motif linking the TCR α chain and TCR β chaintogether.

In some embodiments, the TCR is a scTCR. Typically, a scTCR can begenerated using methods known, See e.g., Soo Hoo, W. F. et al. PNAS(USA) 89, 4759 (1992); Wulfing, C. and P10ckthun, A., J. Mol. Biol. 242,655 (1994); Kurucz, I. et al. PNAS (USA) 90 3830 (1993); Internationalpublished PCT Nos. WO 96/13593, WO 96/18105, WO99/60120, WO99/18129, WO03/020763, WO2011/044186; and Schlueter, C. J. et al. J. Mol. Biol. 256,859 (1996). In some embodiments, a scTCR contains an introducednon-native disulfide interchain bond to facilitate the association ofthe TCR chains (see e.g. International published PCT No. WO 03/020763).In some embodiments, a scTCR is a non-disulfide linked truncated TCR inwhich heterologous leucine zippers fused to the C-termini thereoffacilitate chain association (see e.g. International published PCT No.WO99/60120). In some embodiments, a scTCR contain a TCRα variable domaincovalently linked to a TCRP variable domain via a peptide linker (seee.g., International published PCT No. WO99/18129).

In some embodiments, a scTCR contains a first segment constituted by anamino acid sequence corresponding to a TCR α chain variable region, asecond segment constituted by an amino acid sequence corresponding to aTCR β chain variable region sequence fused to the N terminus of an aminoacid sequence corresponding to a TCR β chain constant domainextracellular sequence, and a linker sequence linking the C terminus ofthe first segment to the N terminus of the second segment.

In some embodiments, a scTCR contains a first segment constituted by ana chain variable region sequence fused to the N terminus of an a chainextracellular constant domain sequence, and a second segment constitutedby a chain variable region sequence fused to the N terminus of asequence Rchain extracellular constant and transmembrane sequence, and,optionally, a linker sequence linking the C terminus of the firstsegment to the N terminus of the second segment.

In some embodiments, a scTCR contains a first segment constituted by aTCR R chain variable region sequence fused to the N terminus of a chainextracellular constant domain sequence, and a second segment constitutedby an a chain variable region sequence fused to the N terminus of asequence a chain extracellular constant and transmembrane sequence, and,optionally, a linker sequence linking the C terminus of the firstsegment to the N terminus of the second segment.

In some embodiments, the linker of a scTCRs that links the first andsecond TCR segments can be any linker capable of forming a singlepolypeptide strand, while retaining TCR binding specificity. In someembodiments, the linker sequence may, for example, have the formula-P-AA-P- wherein P is proline and AA represents an amino acid sequencewherein the amino acids are glycine and serine. In some embodiments, thefirst and second segments are paired so that the variable regionsequences thereof are orientated for such binding. Hence, in some cases,the linker has a sufficient length to span the distance between the Cterminus of the first segment and the N terminus of the second segment,or vice versa, but is not too long to block or reduces bonding of thescTCR to the target ligand. In some embodiments, the linker can containfrom or from about 10 to 45 amino acids, such as 10 to 30 amino acids or26 to 41 amino acids residues, for example 29, 30, 31 or 32 amino acids.In some embodiments, the linker has the formula -PGGG-(SGGGG)5-P-wherein P is proline, G is glycine and S is serine (SEQ ID NO:22). Insome embodiments, the linker has the sequence GSADDAKKDAAKKDGKS (SEQ IDNO:23)

In some embodiments, the scTCR contains a covalent disulfide bondlinking a residue of the immunoglobulin region of the constant domain ofthe a chain to a residue of the immunoglobulin region of the constantdomain of the Rchain. In some embodiments, the interchain disulfide bondin a native TCR is not present. For example, in some embodiments, one ormore cysteines can be incorporated into the constant regionextracellular sequences of the first and second segments of the scTCRpolypeptide. In some cases, both a native and a non-native disulfidebond may be desirable.

In some embodiments of a dTCR or scTCR containing introduced interchaindisulfide bonds, the native disulfide bonds are not present. In someembodiments, the one or more of the native cysteines forming a nativeinterchain disulfide bonds are substituted to another residue, such asto a serine or alanine. In some embodiments, an introduced disulfidebond can be formed by mutating non-cysteine residues on the first andsecond segments to cysteine. Exemplary non-native disulfide bonds of aTCR are described in published International PCT No. WO2006/000830.

In some embodiments, the TCR or antigen-binding fragment thereofexhibits an affinity with an equilibrium binding constant for a targetantigen of between or between about 10-5 and 10-12 M and all individualvalues and ranges therein. In some embodiments, the target antigen is anMHC-peptide complex or ligand.

In some embodiments, nucleic acid or nucleic acids encoding a TCR, suchas a and R chains, can be amplified by PCR, cloning or other suitablemeans and cloned into a suitable expression vector or vectors. Theexpression vector can be any suitable recombinant expression vector, andcan be used to transform or transfect any suitable host. Suitablevectors include those designed for propagation and expansion or forexpression or both, such as plasmids and viruses.

In some embodiments, the vector can a vector of the pUC series(Fermentas Life Sciences), the pBluescript series (Stratagene, LaJolla,Calif.), the pET series (Novagen, Madison, Wis.), the pGEX series(Pharmacia Biotech, Uppsala, Sweden), or the pEX series (Clontech, PaloAlto, Calif.). In some cases, bacteriophage vectors, such as XG10,XGT11, λZapII (Stratagene), λEMBL4, and λNM1149, also can be used. Insome embodiments, plant expression vectors can be used and includepBI01, pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech). In someembodiments, animal expression vectors include pEUK-Cl, pMAM and pMAMneo(Clontech). In some embodiments, a viral vector is used, such as aretroviral vector.

In some embodiments, the recombinant expression vectors can be preparedusing standard recombinant DNA techniques. In some embodiments, vectorscan contain regulatory sequences, such as transcription and translationinitiation and termination codons, which are specific to the type ofhost (e.g., bacterium, fungus, plant, or animal) into which the vectoris to be introduced, as appropriate and taking into considerationwhether the vector is DNA- or RNA-based. In some embodiments, the vectorcan contain a nonnative promoter operably linked to the nucleotidesequence encoding the TCR or antigen-binding portion (or otherMHC-peptide binding molecule). In some embodiments, the promoter can bea non-viral promoter or a viral promoter, such as a cytomegalovirus(CMV) promoter, an SV40 promoter, an RSV promoter, and a promoter foundin the long-terminal repeat of the murine stem cell virus. Other knownpromoters also are contemplated.

In some embodiments, to generate a vector encoding a TCR, the α and βchains are PCR amplified from total cDNA isolated from a T cell cloneexpressing the TCR of interest and cloned into an expression vector. Insome embodiments, the a and β chains are cloned into the same vector. Insome embodiments, the α and β chains are cloned into different vectors.In some embodiments, the generated α and β chains are incorporated intoa retroviral, e.g. lentiviral, vector. Genetically Engineered Cells andMethods of Producing Cells

In some embodiments, the provided methods involve administering to asubject having a disease or condition cells expressing a recombinantantigen receptor. Various methods for the introduction of geneticallyengineered components, e.g., recombinant receptors, e.g., CARs or TCRs,are well known and may be used with the provided methods andcompositions. Exemplary methods include those for transfer of nucleicacids encoding the receptors, including via viral, e.g., retroviral orlentiviral, transduction, transposons, and electroporation.

Among the cells expressing the receptors and administered by theprovided methods are engineered cells. The genetic engineering generallyinvolves introduction of a nucleic acid encoding the recombinant orengineered component into a composition containing the cells, such as byretroviral transduction, transfection, or transformation.

C. Vectors and Methods for Genetic Engineering

Also provided are one or more polynucleotides (e.g., nucleic acidmolecules) encoding recombinant receptors, vectors for geneticallyengineering cells to express the receptors and methods for producing theengineered cells. In some aspects, the recombinant receptor is orcontains a chimeric antigen receptor (CAR). In some aspects, therecombinant receptor is or contains a T cell receptor (TCR), e.g., atransgenic TCR.

In some cases, the nucleic acid sequence encoding the recombinantreceptor, e.g., chimeric antigen receptor (CAR) contains a signalsequence that encodes a signal peptide. Non-limiting exemplary examplesof signal peptides include, for example, the GMCSFR alpha chain signalpeptide set forth in SEQ ID NO: 26 and encoded by the nucleotidesequence set forth in SEQ ID NO: 25, the CD8 alpha signal peptide setforth in SEQ ID NO: 24, or the CD33 signal peptide set forth in SEQ IDNO:60.

In some embodiments, recombinant nucleic acids are transferred intocells using recombinant infectious virus particles, such as, e.g.,vectors derived from simian virus 40 (SV40), adenoviruses,adeno-associated virus (AAV). In some embodiments, recombinant nucleicacids are transferred into T cells using recombinant lentiviral vectorsor retroviral vectors, such as gamma-retroviral vectors (see, e.g.,Koste et al. (2014) Gene Therapy 2014 Apr. 3. doi: 10.1038/gt.2014.25;Carlens et al. (2000) Exp Hematol 28(10): 1137-46; Alonso-Camino et al.(2013) Mol Ther Nucl Acids 2, e93; Park et al., Trends Biotechnol. 2011Nov. 29(11): 550-557.

In some embodiments, the retroviral vector has a long terminal repeatsequence (LTR), e.g., a retroviral vector derived from the Moloneymurine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV),murine embryonic stem cell virus (MESV), murine stem cell virus (MSCV),spleen focus forming virus (SFFV), or adeno-associated virus (AAV). Mostretroviral vectors are derived from murine retroviruses. In someembodiments, the retroviruses include those derived from any avian ormammalian cell source. The retroviruses typically are amphotropic,meaning that they are capable of infecting host cells of severalspecies, including humans. In one embodiment, the gene to be expressedreplaces the retroviral gag, pol and/or env sequences. A number ofillustrative retroviral systems have been described (e.g., U.S. Pat.Nos. 5,219,740; 6,207,453; 5,219,740; Miller and Rosman (1989)BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy 1:5-14;Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993) Proc.Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993)Cur. Opin. Genet. Develop. 3:102-109.

Methods of lentiviral transduction are known. Exemplary methods aredescribed in, e.g., Wang et al. (2012) J. Immunother. 35(9): 689-701;Cooper et al. (2003) Blood. 101:1637-1644; Verhoeyen et al. (2009)Methods Mol Biol. 506: 97-114; and Cavalieri et al. (2003) Blood.102(2): 497-505.

In some embodiments, recombinant nucleic acids are transferred into Tcells via electroporation (see, e.g., Chicaybam et al, (2013) PLoS ONE8(3): e60298 and Van Tedeloo et al. (2000) Gene Therapy 7(16):1431-1437). In some embodiments, recombinant nucleic acids aretransferred into T cells via transposition (see, e.g., Manuri et al.(2010) Hum Gene Ther 21(4): 427-437; Sharma et al. (2013) Molec TherNucl Acids 2, e74; and Huang et al. (2009) Methods Mol Biol 506:115-126). Other methods of introducing and expressing genetic materialin immune cells include calcium phosphate transfection (e.g., asdescribed in Current Protocols in Molecular Biology, John Wiley & Sons,New York. N.Y.), protoplast fusion, cationic liposome-mediatedtransfection; tungsten particle-facilitated microparticle bombardment(Johnston, Nature, 346: 776-777 (1990)); and strontium phosphate DNAco-precipitation (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)).

Other approaches and vectors for transfer of the nucleic acids encodingthe recombinant products are those described, e.g., in internationalpatent application, Publication No.: WO2014055668, and U.S. Pat. No.7,446,190.

In some embodiments, the cells, e.g., T cells, may be transfected eitherduring or after expansion e.g. with a T cell receptor (TCR) or achimeric antigen receptor (CAR). This transfection for the introductionof the gene of the desired receptor can be carried out with any suitableretroviral vector, for example. The genetically modified cell populationcan then be liberated from the initial stimulus (the anti-CD3/anti-CD28stimulus, for example) and subsequently be stimulated with a second typeof stimulus e.g. via a de novo introduced receptor). This second type ofstimulus may include an antigenic stimulus in form of a peptide/MHCmolecule, the cognate (cross-linking) ligand of the geneticallyintroduced receptor (e.g. natural ligand of a CAR) or any ligand (suchas an antibody) that directly binds within the framework of the newreceptor (e.g. by recognizing constant regions within the receptor).See, for example, Cheadle et al, “Chimeric antigen receptors for T-cellbased therapy” Methods Mol Biol. 2012; 907:645-66 or Barrett et al.,Chimeric Antigen Receptor Therapy for Cancer Annual Review of MedicineVol. 65: 333-347 (2014).

In some cases, a vector may be used that does not require that thecells, e.g., T cells, are activated. In some such instances, the cellsmay be selected and/or transduced prior to activation. Thus, the cellsmay be engineered prior to, or subsequent to culturing of the cells, andin some cases at the same time as or during at least a portion of theculturing.

Among additional nucleic acids, e.g., genes for introduction are thoseto improve the efficacy of therapy, such as by promoting viabilityand/or function of transferred cells; genes to provide a genetic markerfor selection and/or evaluation of the cells, such as to assess in vivosurvival or localization; genes to improve safety, for example, bymaking the cell susceptible to negative selection in vivo as describedby Lupton S. D. et al., Mol. and Cell Biol., 11:6 (1991); and Riddell etal., Human Gene Therapy 3:319-338 (1992); see also the publications ofPCT/US91/08442 and PCT/US94/05601 by Lupton et al. describing the use ofbifunctional selectable fusion genes derived from fusing a dominantpositive selectable marker with a negative selectable marker. See, e.g.,Riddell et al., U.S. Pat. No. 6,040,177, at columns 14-17.

D. Cells and Preparation of Cells for Genetic Engineering

In some embodiments, the nucleic acids are heterologous, i.e., normallynot present in a cell or sample obtained from the cell, such as oneobtained from another organism or cell, which for example, is notordinarily found in the cell being engineered and/or an organism fromwhich such cell is derived. In some embodiments, the nucleic acids arenot naturally occurring, such as a nucleic acid not found in nature,including one comprising chimeric combinations of nucleic acids encodingvarious domains from multiple different cell types.

The cells generally are eukaryotic cells, such as mammalian cells, andtypically are human cells. In some embodiments, the cells are derivedfrom the blood, bone marrow, lymph, or lymphoid organs, are cells of theimmune system, such as cells of the innate or adaptive immunity, e.g.,myeloid or lymphoid cells, including lymphocytes, typically T cellsand/or NK cells. Other exemplary cells include stem cells, such asmultipotent and pluripotent stem cells, including induced pluripotentstem cells (iPSCs). The cells typically are primary cells, such as thoseisolated directly from a subject and/or isolated from a subject andfrozen. In some embodiments, the cells include one or more subsets of Tcells or other cell types, such as whole T cell populations, CD4+ cells,CD8+ cells, and subpopulations thereof, such as those defined byfunction, activation state, maturity, potential for differentiation,expansion, recirculation, localization, and/or persistence capacities,antigen-specificity, type of antigen receptor, presence in a particularorgan or compartment, marker or cytokine secretion profile, and/ordegree of differentiation. With reference to the subject to be treated,the cells may be allogeneic and/or autologous. Among the methods includeoff-the-shelf methods. In some aspects, such as for off-the-shelftechnologies, the cells are pluripotent and/or multipotent, such as stemcells, such as induced pluripotent stem cells (iPSCs). In someembodiments, the methods include isolating cells from the subject,preparing, processing, culturing, and/or engineering them, andre-introducing them into the same subject, before or aftercryopreservation.

Among the sub-types and subpopulations of T cells and/or of CD4+ and/orof CD8+ T cells are naïve T (T_(N)) cells, effector T cells (T_(EFF)),memory T cells and sub-types thereof, such as stem cell memory T(T_(SCM)), central memory T (T_(EFF)), effector memory T (T_(EM)), orterminally differentiated effector memory T cells, tumor-infiltratinglymphocytes (TIL), immature T cells, mature T cells, helper T cells,cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturallyoccurring and adaptive regulatory T (Treg) cells, helper T cells, suchas TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells,follicular helper T cells, alpha/beta T cells, and delta/gamma T cells.

In some embodiments, the cells are natural killer (NK) cells. In someembodiments, the cells are monocytes or granulocytes, e.g., myeloidcells, macrophages, neutrophils, dendritic cells, mast cells,eosinophils, and/or basophils.

In some embodiments, the cells include one or more nucleic acidsintroduced via genetic engineering, and thereby express recombinant orgenetically engineered products of such nucleic acids. In someembodiments, the nucleic acids are heterologous, i.e., normally notpresent in a cell or sample obtained from the cell, such as one obtainedfrom another organism or cell, which for example, is not ordinarilyfound in the cell being engineered and/or an organism from which suchcell is derived. In some embodiments, the nucleic acids are notnaturally occurring, such as a nucleic acid not found in nature,including one comprising chimeric combinations of nucleic acids encodingvarious domains from multiple different cell types.

In some embodiments, preparation of the engineered cells includes one ormore culture and/or preparation steps. The cells for introduction of thenucleic acid encoding the transgenic receptor such as the CAR, may beisolated from a sample, such as a biological sample, e.g., one obtainedfrom or derived from a subject. In some embodiments, the subject fromwhich the cell is isolated is one having the disease or condition or inneed of a cell therapy or to which cell therapy will be administered.The subject in some embodiments is a human in need of a particulartherapeutic intervention, such as the adoptive cell therapy for whichcells are being isolated, processed, and/or engineered.

Accordingly, the cells in some embodiments are primary cells, e.g.,primary human cells. The samples include tissue, fluid, and othersamples taken directly from the subject, as well as samples resultingfrom one or more processing steps, such as separation, centrifugation,genetic engineering (e.g. transduction with viral vector), washing,and/or incubation. The biological sample can be a sample obtaineddirectly from a biological source or a sample that is processed.Biological samples include, but are not limited to, body fluids, such asblood, plasma, serum, cerebrospinal fluid, synovial fluid, urine andsweat, tissue and organ samples, including processed samples derivedtherefrom.

In some aspects, the sample from which the cells are derived or isolatedis blood or a blood-derived sample, or is or is derived from anapheresis or leukapheresis product. Exemplary samples include wholeblood, peripheral blood mononuclear cells (PBMCs), leukocytes, bonemarrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node,gut associated lymphoid tissue, mucosa associated lymphoid tissue,spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon,kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries,tonsil, or other organ, and/or cells derived therefrom. Samples include,in the context of cell therapy, e.g., adoptive cell therapy, samplesfrom autologous and allogeneic sources.

In some embodiments, the cells are derived from cell lines, e.g., T celllines. The cells in some embodiments are obtained from a xenogeneicsource, for example, from mouse, rat, non-human primate, and pig.

In some embodiments, isolation of the cells includes one or morepreparation and/or non-affinity based cell separation steps. In someexamples, cells are washed, centrifuged, and/or incubated in thepresence of one or more reagents, for example, to remove unwantedcomponents, enrich for desired components, lyse or remove cellssensitive to particular reagents. In some examples, cells are separatedbased on one or more property, such as density, adherent properties,size, sensitivity and/or resistance to particular components.

In some examples, cells from the circulating blood of a subject areobtained, e.g., by apheresis or leukapheresis. The samples, in someaspects, contain lymphocytes, including T cells, monocytes,granulocytes, B cells, other nucleated white blood cells, red bloodcells, and/or platelets, and in some aspects contains cells other thanred blood cells and platelets.

In some embodiments, the blood cells collected from the subject arewashed, e.g., to remove the plasma fraction and to place the cells in anappropriate buffer or media for subsequent processing steps. In someembodiments, the cells are washed with phosphate buffered saline (PBS).In some embodiments, the wash solution lacks calcium and/or magnesiumand/or many or all divalent cations. In some aspects, a washing step isaccomplished a semi-automated “flow-through” centrifuge (for example,the Cobe 2991 cell processor, Baxter) according to the manufacturer'sinstructions. In some aspects, a washing step is accomplished bytangential flow filtration (TFF) according to the manufacturer'sinstructions. In some embodiments, the cells are resuspended in avariety of biocompatible buffers after washing, such as, for example,Ca⁺⁺/Mg⁺⁺ free PBS. In certain embodiments, components of a blood cellsample are removed and the cells directly resuspended in culture media.

In some embodiments, the methods include density-based cell separationmethods, such as the preparation of white blood cells from peripheralblood by lysing the red blood cells and centrifugation through a Percollor Ficoll gradient.

In some embodiments, the isolation methods include the separation ofdifferent cell types based on the expression or presence in the cell ofone or more specific molecules, such as surface markers, e.g., surfaceproteins, intracellular markers, or nucleic acid. In some embodiments,any known method for separation based on such markers may be used. Insome embodiments, the separation is affinity- or immunoaffinity-basedseparation. For example, the isolation in some aspects includesseparation of cells and cell populations based on the cells' expressionor expression level of one or more markers, typically cell surfacemarkers, for example, by incubation with an antibody or binding partnerthat specifically binds to such markers, followed generally by washingsteps and separation of cells having bound the antibody or bindingpartner, from those cells having not bound to the antibody or bindingpartner.

Such separation steps can be based on positive selection, in which thecells having bound the reagents are retained for further use, and/ornegative selection, in which the cells having not bound to the antibodyor binding partner are retained. In some examples, both fractions areretained for further use. In some aspects, negative selection can beparticularly useful where no antibody is available that specificallyidentifies a cell type in a heterogeneous population, such thatseparation is best carried out based on markers expressed by cells otherthan the desired population.

The separation need not result in 100% enrichment or removal of aparticular cell population or cells expressing a particular marker. Forexample, positive selection of or enrichment for cells of a particulartype, such as those expressing a marker, refers to increasing the numberor percentage of such cells, but need not result in a complete absenceof cells not expressing the marker. Likewise, negative selection,removal, or depletion of cells of a particular type, such as thoseexpressing a marker, refers to decreasing the number or percentage ofsuch cells, but need not result in a complete removal of all such cells.

In some examples, multiple rounds of separation steps are carried out,where the positively or negatively selected fraction from one step issubjected to another separation step, such as a subsequent positive ornegative selection. In some examples, a single separation step candeplete cells expressing multiple markers simultaneously, such as byincubating cells with a plurality of antibodies or binding partners,each specific for a marker targeted for negative selection. Likewise,multiple cell types can simultaneously be positively selected byincubating cells with a plurality of antibodies or binding partnersexpressed on the various cell types.

For example, in some aspects, specific subpopulations of T cells, suchas cells positive or expressing high levels of one or more surfacemarkers, e.g., CD28⁺, CD62L⁺, CCR7⁺, CD27⁺, CD127⁺, CD4⁺, CD8⁺, CD45RA⁺,and/or CD45RO⁺ T cells, are isolated by positive or negative selectiontechniques.

For example, CD3+, CD28⁺ T cells can be positively selected usinganti-CD3/anti-CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450CD3/CD28 T Cell Expander).

In some embodiments, isolation is carried out by enrichment for aparticular cell population by positive selection, or depletion of aparticular cell population, by negative selection. In some embodiments,positive or negative selection is accomplished by incubating cells withone or more antibodies or other binding agent that specifically bind toone or more surface markers expressed or expressed (marker⁺) at arelatively higher level (marker^(high)) on the positively or negativelyselected cells, respectively.

In some embodiments, T cells are separated from a PBMC sample bynegative selection of markers expressed on non-T cells, such as B cells,monocytes, or other white blood cells, such as CD14. In some aspects, aCD4⁺ or CD8⁺ selection step is used to separate CD4⁺ helper and CD8⁺cytotoxic T cells. Such CD4⁺ and CD8⁺ populations can be further sortedinto sub-populations by positive or negative selection for markersexpressed or expressed to a relatively higher degree on one or morenaive, memory, and/or effector T cell subpopulations.

In some embodiments, CD8⁺ cells are further enriched for or depleted ofnaive, central memory, effector memory, and/or central memory stemcells, such as by positive or negative selection based on surfaceantigens associated with the respective subpopulation. In someembodiments, enrichment for central memory T (T_(CM)) cells is carriedout to increase efficacy, such as to improve long-term survival,expansion, and/or engraftment following administration, which in someaspects is particularly robust in such sub-populations. See Terakura etal. (2012) Blood. 1:72-82; Wang et al. (2012) J Immunother.35(9):689-701. In some embodiments, combining T_(CM)-enriched CD8⁺ Tcells and CD4⁺ T cells further enhances efficacy.

In embodiments, memory T cells are present in both CD62L⁺ and CD62L⁻subsets of CD8⁺ peripheral blood lymphocytes. PBMC can be enriched foror depleted of CD62L⁻CD8⁺ and/or CD62L⁺CD8⁺ fractions, such as usinganti-CD8 and anti-CD62L antibodies.

In some embodiments, the enrichment for central memory T (T_(CM)) cellsis based on positive or high surface expression of CD45RO, CD62L, CCR7,CD28, CD3, and/or CD127; in some aspects, it is based on negativeselection for cells expressing or highly expressing CD45RA and/orgranzyme B. In some aspects, isolation of a CD8⁺ population enriched forT_(CM) cells is carried out by depletion of cells expressing CD4, CD14,CD45RA, and positive selection or enrichment for cells expressing CD62L.In one aspect, enrichment for central memory T (T_(CM)) cells is carriedout starting with a negative fraction of cells selected based on CD4expression, which is subjected to a negative selection based onexpression of CD14 and CD45RA, and a positive selection based on CD62L.Such selections in some aspects are carried out simultaneously and inother aspects are carried out sequentially, in either order. In someaspects, the same CD4 expression-based selection step used in preparingthe CD8⁺ cell population or subpopulation, also is used to generate theCD4⁺ cell population or subpopulation, such that both the positive andnegative fractions from the CD4-based separation are retained and usedin subsequent steps of the methods, optionally following one or morefurther positive or negative selection steps.

In a particular example, a sample of PBMCs or other white blood cellsample is subjected to selection of CD4⁺ cells, where both the negativeand positive fractions are retained. The negative fraction then issubjected to negative selection based on expression of CD14 and CD45RAor CD19, and positive selection based on a marker characteristic ofcentral memory T cells, such as CD62L or CCR7, where the positive andnegative selections are carried out in either order.

CD4⁺ T helper cells are sorted into naïve, central memory, and effectorcells by identifying cell populations that have cell surface antigens.CD4⁺ lymphocytes can be obtained by standard methods. In someembodiments, naive CD4⁺ T lymphocytes are CD45RO⁻, CD45RA⁺, CD62L+, CD4⁺T cells. In some embodiments, central memory CD4⁺ cells are CD62L⁺ andCD45RO⁺. In some embodiments, effector CD4⁺ cells are CD62L⁻ andCD45RO⁻.

In one example, to enrich for CD4⁺ cells by negative selection, amonoclonal antibody cocktail typically includes antibodies to CD14,CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, the antibody orbinding partner is bound to a solid support or matrix, such as amagnetic bead or paramagnetic bead, to allow for separation of cells forpositive and/or negative selection. For example, in some embodiments,the cells and cell populations are separated or isolated usingimmunomagnetic (or affinitymagnetic) separation techniques (reviewed inMethods in Molecular Medicine, vol. 58: Metastasis Research Protocols,Vol. 2: Cell Behavior In Vitro and In Vivo, p 17-25 Edited by: S. A.Brooks and U. Schumacher © Humana Press Inc., Totowa, N.J.).

In some aspects, the sample or composition of cells to be separated isincubated with small, magnetizable or magnetically responsive material,such as magnetically responsive particles or microparticles, such asparamagnetic beads (e.g., such as Dynalbeads or MACS beads). Themagnetically responsive material, e.g., particle, generally is directlyor indirectly attached to a binding partner, e.g., an antibody, thatspecifically binds to a molecule, e.g., surface marker, present on thecell, cells, or population of cells that it is desired to separate,e.g., that it is desired to negatively or positively select.

In some embodiments, the magnetic particle or bead comprises amagnetically responsive material bound to a specific binding member,such as an antibody or other binding partner. There are many well-knownmagnetically responsive materials used in magnetic separation methods.Suitable magnetic particles include those described in Molday, U.S. Pat.No. 4,452,773, and in European Patent Specification EP 452342 B, whichare hereby incorporated by reference. Colloidal sized particles, such asthose described in Owen U.S. Pat. No. 4,795,698, and Liberti et al.,U.S. Pat. No. 5,200,084 are other examples.

The incubation generally is carried out under conditions whereby theantibodies or binding partners, or molecules, such as secondaryantibodies or other reagents, which specifically bind to such antibodiesor binding partners, which are attached to the magnetic particle orbead, specifically bind to cell surface molecules if present on cellswithin the sample.

In some aspects, the sample is placed in a magnetic field, and thosecells having magnetically responsive or magnetizable particles attachedthereto will be attracted to the magnet and separated from the unlabeledcells. For positive selection, cells that are attracted to the magnetare retained; for negative selection, cells that are not attracted(unlabeled cells) are retained. In some aspects, a combination ofpositive and negative selection is performed during the same selectionstep, where the positive and negative fractions are retained and furtherprocessed or subject to further separation steps.

In certain embodiments, the magnetically responsive particles are coatedin primary antibodies or other binding partners, secondary antibodies,lectins, enzymes, or streptavidin. In certain embodiments, the magneticparticles are attached to cells via a coating of primary antibodiesspecific for one or more markers. In certain embodiments, the cells,rather than the beads, are labeled with a primary antibody or bindingpartner, and then cell-type specific secondary antibody- or otherbinding partner (e.g., streptavidin)-coated magnetic particles, areadded. In certain embodiments, streptavidin-coated magnetic particlesare used in conjunction with biotinylated primary or secondaryantibodies.

In some embodiments, the magnetically responsive particles are leftattached to the cells that are to be subsequently incubated, culturedand/or engineered; in some aspects, the particles are left attached tothe cells for administration to a patient. In some embodiments, themagnetizable or magnetically responsive particles are removed from thecells. Methods for removing magnetizable particles from cells are knownand include, e.g., the use of competing non-labeled antibodies, andmagnetizable particles or antibodies conjugated to cleavable linkers. Insome embodiments, the magnetizable particles are biodegradable.

In some embodiments, the affinity-based selection is viamagnetic-activated cell sorting (MACS) (Miltenyi Biotec, Auburn,Calif.). Magnetic Activated Cell Sorting (MACS) systems are capable ofhigh-purity selection of cells having magnetized particles attachedthereto. In certain embodiments, MACS operates in a mode wherein thenon-target and target species are sequentially eluted after theapplication of the external magnetic field. That is, the cells attachedto magnetized particles are held in place while the unattached speciesare eluted. Then, after this first elution step is completed, thespecies that were trapped in the magnetic field and were prevented frombeing eluted are freed in some manner such that they can be eluted andrecovered. In certain embodiments, the non-target cells are labelled anddepleted from the heterogeneous population of cells.

In certain embodiments, the isolation or separation is carried out usinga system, device, or apparatus that carries out one or more of theisolation, cell preparation, separation, processing, incubation,culture, and/or formulation steps of the methods. In some aspects, thesystem is used to carry out each of these steps in a closed or sterileenvironment, for example, to minimize error, user handling and/orcontamination. In one example, the system is a system as described inInternational Patent Application, Publication Number WO2009/072003, orUS 20110003380 A1.

In some embodiments, the system or apparatus carries out one or more,e.g., all, of the isolation, processing, engineering, and formulationsteps in an integrated or self-contained system, and/or in an automatedor programmable fashion. In some aspects, the system or apparatusincludes a computer and/or computer program in communication with thesystem or apparatus, which allows a user to program, control, assess theoutcome of, and/or adjust various aspects of the processing, isolation,engineering, and formulation steps.

In some aspects, the separation and/or other steps is carried out usingCliniMACS system (Miltenyi Biotec), for example, for automatedseparation of cells on a clinical-scale level in a closed and sterilesystem. Components can include an integrated microcomputer, magneticseparation unit, peristaltic pump, and various pinch valves. Theintegrated computer in some aspects controls all components of theinstrument and directs the system to perform repeated procedures in astandardized sequence. The magnetic separation unit in some aspectsincludes a movable permanent magnet and a holder for the selectioncolumn. The peristaltic pump controls the flow rate throughout thetubing set and, together with the pinch valves, ensures the controlledflow of buffer through the system and continual suspension of cells.

The CliniMACS system in some aspects uses antibody-coupled magnetizableparticles that are supplied in a sterile, non-pyrogenic solution. Insome embodiments, after labelling of cells with magnetic particles thecells are washed to remove excess particles. A cell preparation bag isthen connected to the tubing set, which in turn is connected to a bagcontaining buffer and a cell collection bag. The tubing set consists ofpre-assembled sterile tubing, including a pre-column and a separationcolumn, and are for single use only. After initiation of the separationprogram, the system automatically applies the cell sample onto theseparation column. Labelled cells are retained within the column, whileunlabeled cells are removed by a series of washing steps. In someembodiments, the cell populations for use with the methods describedherein are unlabeled and are not retained in the column. In someembodiments, the cell populations for use with the methods describedherein are labeled and are retained in the column. In some embodiments,the cell populations for use with the methods described herein areeluted from the column after removal of the magnetic field, and arecollected within the cell collection bag.

In certain embodiments, separation and/or other steps are carried outusing the CliniMACS Prodigy system (Miltenyi Biotec). The CliniMACSProdigy system in some aspects is equipped with a cell processing unitythat permits automated washing and fractionation of cells bycentrifugation. The CliniMACS Prodigy system can also include an onboardcamera and image recognition software that determines the optimal cellfractionation endpoint by discerning the macroscopic layers of thesource cell product. For example, peripheral blood is automaticallyseparated into erythrocytes, white blood cells and plasma layers. TheCliniMACS Prodigy system can also include an integrated cell cultivationchamber which accomplishes cell culture protocols such as, e.g., celldifferentiation and expansion, antigen loading, and long-term cellculture. Input ports can allow for the sterile removal and replenishmentof media and cells can be monitored using an integrated microscope. See,e.g., Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura etal. (2012) Blood. 1:72-82, and Wang et al. (2012) J Immunother.35(9):689-701.

In some embodiments, a cell population described herein is collected andenriched (or depleted) via flow cytometry, in which cells stained formultiple cell surface markers are carried in a fluidic stream. In someembodiments, a cell population described herein is collected andenriched (or depleted) via preparative scale (FACS)-sorting. In certainembodiments, a cell population described herein is collected andenriched (or depleted) by use of microelectromechanical systems (MEMS)chips in combination with a FACS-based detection system (see, e.g., WO2010/033140, Cho et al. (2010) Lab Chip 10, 1567-1573; and Godin et al.(2008) J Biophoton. 1(5):355-376. In both cases, cells can be labeledwith multiple markers, allowing for the isolation of well-defined T cellsubsets at high purity.

In some embodiments, the antibodies or binding partners are labeled withone or more detectable marker, to facilitate separation for positiveand/or negative selection. For example, separation may be based onbinding to fluorescently labeled antibodies. In some examples,separation of cells based on binding of antibodies or other bindingpartners specific for one or more cell surface markers are carried in afluidic stream, such as by fluorescence-activated cell sorting (FACS),including preparative scale (FACS) and/or microelectromechanical systems(MEMS) chips, e.g., in combination with a flow-cytometric detectionsystem. Such methods allow for positive and negative selection based onmultiple markers simultaneously.

In some embodiments, the preparation methods include steps for freezing,e.g., cryopreserving, the cells, either before or after isolation,incubation, and/or engineering. In some embodiments, the freeze andsubsequent thaw step removes granulocytes and, to some extent, monocytesin the cell population. In some embodiments, the cells are suspended ina freezing solution, e.g., following a washing step to remove plasma andplatelets. Any of a variety of known freezing solutions and parametersin some aspects may be used. One example involves using PBS containing20% DMSO and 8% human serum albumin (HSA), or other suitable cellfreezing media. This is then diluted 1:1 with media so that the finalconcentration of DMSO and HSA are 10% and 4%, respectively. The cellsare generally then frozen to −80° C. at a rate of 1° per minute andstored in the vapor phase of a liquid nitrogen storage tank.

In some embodiments, the cells are incubated and/or cultured prior to orin connection with genetic engineering. The incubation steps can includeculture, cultivation, stimulation, activation, and/or propagation. Theincubation and/or engineering may be carried out in a culture vessel,such as a unit, chamber, well, column, tube, tubing set, valve, vial,culture dish, bag, or other container for culture or cultivating cells.In some embodiments, the compositions or cells are incubated in thepresence of stimulating conditions or a stimulatory agent. Suchconditions include those designed to induce proliferation, expansion,activation, and/or survival of cells in the population, to mimic antigenexposure, and/or to prime the cells for genetic engineering, such as forthe introduction of a recombinant antigen receptor.

The conditions can include one or more of particular media, temperature,oxygen content, carbon dioxide content, time, agents, e.g., nutrients,amino acids, antibiotics, ions, and/or stimulatory factors, such ascytokines, chemokines, antigens, binding partners, fusion proteins,recombinant soluble receptors, and any other agents designed to activatethe cells.

In some embodiments, the stimulating conditions or agents include one ormore agent, e.g., ligand, which is capable of activating anintracellular signaling domain of a TCR complex. In some aspects, theagent turns on or initiates TCR/CD3 intracellular signaling cascade in aT cell. Such agents can include antibodies, such as those specific for aTCR, e.g. anti-CD3. In some embodiments, the stimulating conditionsinclude one or more agent, e.g. ligand, which is capable of stimulatinga costimulatory receptor, e.g., anti-CD28. In some embodiments, suchagents and/or ligands may be, bound to solid support such as a bead,and/or one or more cytokines. Optionally, the expansion method mayfurther comprise the step of adding anti-CD3 and/or anti-CD28 antibodyto the culture medium (e.g., at a concentration of at least about 0.5ng/ml). In some embodiments, the stimulating agents include IL-2, IL-15and/or IL-7. In some aspects, the IL-2 concentration is at least about10 units/mL.

In some aspects, incubation is carried out in accordance with techniquessuch as those described in U.S. Pat. No. 6,040,177 to Riddell et al.,Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura et al.(2012) Blood. 1:72-82, and/or Wang et al. (2012) J Immunother.35(9):689-701.

In some embodiments, the T cells are expanded by adding to aculture-initiating composition feeder cells, such as non-dividingperipheral blood mononuclear cells (PBMC), (e.g., such that theresulting population of cells contains at least about 5, 10, 20, or 40or more PBMC feeder cells for each T lymphocyte in the initialpopulation to be expanded); and incubating the culture (e.g. for a timesufficient to expand the numbers of T cells). In some aspects, thenon-dividing feeder cells can comprise gamma-irradiated PBMC feedercells. In some embodiments, the PBMC are irradiated with gamma rays inthe range of about 3000 to 3600 rads to prevent cell division. In someaspects, the feeder cells are added to culture medium prior to theaddition of the populations of T cells.

In some embodiments, the stimulating conditions include temperaturesuitable for the growth of human T lymphocytes, for example, at leastabout 25 degrees Celsius, generally at least about 30 degrees, andgenerally at or about 37 degrees Celsius. Optionally, the incubation mayfurther comprise adding non-dividing EBV-transformed lymphoblastoidcells (LCL) as feeder cells. LCL can be irradiated with gamma rays inthe range of about 6000 to 10,000 rads. The LCL feeder cells in someaspects is provided in any suitable amount, such as a ratio of LCLfeeder cells to initial T lymphocytes of at least about 10:1.

In embodiments, antigen-specific T cells, such as antigen-specific CD4+and/or CD8+ T cells, are obtained by stimulating naive or antigenspecific T lymphocytes with antigen. For example, antigen-specific Tcell lines or clones can be generated to cytomegalovirus antigens byisolating T cells from infected subjects and stimulating the cells invitro with the same antigen.

E. Administration of Cell Therapy

Provided herein are methods to assess, predict, infer, and/or estimate arisk of toxicity, for example following administration of a therapy suchas a cell therapy. In some embodiments, the subject is administered,will be administered, or is a candidate to be administered a therapy,e.g., an immunotherapy and/or a cell therapy. Further provided hereinare methods of administering a cell therapy, such as methods ofselecting a subject and administering a dose of cell therapy, whereinthe subject is selected based on the risk, probability, and/orlikelihood of toxicity.

In certain embodiments, the methods include steps to assess, determine,measure, and/or quantify a risk, probability, and/or likelihood that asubject will experience and/or develop a toxicity followingadministration of or associated with a cell therapy. In someembodiments, the subject's risk, probability, and/or likelihood ofexperiencing or developing the toxicity is assessed, determined,measured and/or quantified by a method of assessing a factor indicativeof tumor burden, such as described herein. In particular embodiments,the subject is determined to have a low, reduced, and/or decreased risk,probability and/or likelihood of toxicity, and the subject isadministered a standard dose of a cell therapy. In certain embodiments,the subject is determined to have a high, elevated, or increased risk oftoxicity, and the subject is administered a dose of a cell therapy thatis less than a standard dose of the cell therapy. In particularembodiments, the subject is determined to have a high, elevated, orincreased risk of toxicity, and the subject is administered a standarddose of a cell therapy and an intervention, such as any as describedherein. In certain embodiments, the subject is determined to have ahigh, elevated, or increased risk of toxicity, and the subject isadministered a dose of a cell therapy that is lower than the standarddose of the cell therapy and an intervention, such as any as describedherein.

1. Methods of Treatment

In certain embodiments of the methods provided herein, the subject isadministered a standard dose of a therapy, e.g., an immunotherapy or acell therapy. In certain embodiments, the risk, probability, and/orlikelihood that the subject will experience and/or develop a toxicityfollowing administration of or associated with the cell therapy isdetermined by one or more methods of assessing a factor indicative oftumor burden, such as provided herein, e.g. in Section I. In certainembodiments, the subject is determined to be unlikely, or to have a lowrisk, probability, and/or likelihood, of developing a toxicity, e.g.,CRS or neurotoxicity, by performing one or more methods as described,such as provided in Section I, and the subject is administered astandard dose of the cell therapy. In particular embodiments, thesubject is administered a reduced dose of the therapeutic cellcomposition. In some embodiments, the subject is determined to have ahigh, elevated, and/or an increased risk, probability, and/or likelihoodof developing a toxicity, e.g., a neurotoxicity, and the subject isadministered a reduced dose of the therapeutic cell composition. In someembodiments, the cell therapy is a T cell therapy. In some embodiments,the T cell therapy contains cells that express a recombinant receptor.In some embodiments, the recombinant receptor is a CAR.

The disease or condition that is treated in some aspects can be any inwhich expression of an antigen is associated with, specific to, and/orexpressed on a cell or tissue of a disease, disorder or condition and/orinvolved in the etiology of a disease, condition or disorder, e.g.causes, exacerbates or otherwise is involved in such disease, condition,or disorder. Exemplary diseases and conditions can include diseases orconditions associated with malignancy or transformation of cells (e.g.cancer), autoimmune or inflammatory disease, or an infectious disease,e.g. caused by a bacterial, viral or other pathogen. Exemplary antigens,which include antigens associated with various diseases and conditionsthat can be treated, are described above. In particular embodiments, theimmunomodulatory polypeptide and/or recombinant receptor, e.g., thechimeric antigen receptor or TCR, specifically binds to an antigenassociated with the disease or condition. In some embodiments, thesubject has a disease, disorder or condition, optionally a cancer, atumor, an autoimmune disease, disorder or condition, or an infectiousdisease.

In some embodiments, the disease, disorder or condition includes tumorsassociated with various cancers. The cancer can in some embodiments beany cancer located in the body of a subject, such as, but not limitedto, cancers located at the head and neck, breast, liver, colon, ovary,prostate, pancreas, brain, cervix, bone, skin, eye, bladder, stomach,esophagus, peritoneum, or lung. For example, the anti-cancer agent canbe used for the treatment of colon cancer, cervical cancer, cancer ofthe central nervous system, breast cancer, bladder cancer, analcarcinoma, head and neck cancer, ovarian cancer, endometrial cancer,small cell lung cancer, non-small cell lung carcinoma, neuroendocrinecancer, soft tissue carcinoma, penile cancer, prostate cancer,pancreatic cancer, gastric cancer, gall bladder cancer or espohagealcancer. In some cases, the cancer can be a cancer of the blood. In someembodiments, the disease, disorder or condition is a tumor, such as asolid tumor, lymphoma, leukemia, blood tumor, metastatic tumor, or othercancer or tumor type. In some embodiments, the disease, disorder orcondition is selected from among cancers of the colon, lung, liver,breast, prostate, ovarian, skin, melanoma, bone, brain cancer, ovariancancer, epithelial cancers, renal cell carcinoma, pancreaticadenocarcinoma, cervical carcinoma, colorectal cancer, glioblastoma,neuroblastoma, Ewing sarcoma, medulloblastoma, osteosarcoma, synovialsarcoma, and/or mesothelioma.

Among the diseases, conditions, and disorders are tumors, includingsolid tumors, hematologic malignancies, and melanomas, and includinglocalized and metastatic tumors, infectious diseases, such as infectionwith a virus or other pathogen, e.g., HIV, HCV, HBV, CMV, HPV, andparasitic disease, and autoimmune and inflammatory diseases. In someembodiments, the disease, disorder or condition is a tumor, cancer,malignancy, neoplasm, or other proliferative disease or disorder. Suchdiseases include but are not limited to leukemia, lymphoma, e.g., acutemyeloid (or myelogenous) leukemia (AML), chronic myeloid (ormyelogenous) leukemia (CML), acute lymphocytic (or lymphoblastic)leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia(HCL), small lymphocytic lymphoma (SLL), Mantle cell lymphoma (MCL),Marginal zone lymphoma, Burkitt lymphoma, Hodgkin lymphoma (HL),non-Hodgkin lymphoma (NHL), Anaplastic large cell lymphoma (ALCL),follicular lymphoma, refractory follicular lymphoma, diffuse largeB-cell lymphoma (DLBCL) and multiple myeloma (MM), a B cell malignancyis selected from among acute lymphoblastic leukemia (ALL), adult ALL,chronic lymphoblastic leukemia (CLL), non-Hodgkin lymphoma (NHL), andDiffuse Large B-Cell Lymphoma (DLBCL).

In some embodiments, the disease or condition is an infectious diseaseor condition, such as, but not limited to, viral, retroviral, bacterial,and protozoal infections, immunodeficiency, Cytomegalovirus (CMV),Epstein-Barr virus (EBV), adenovirus, BK polyomavirus. In someembodiments, the disease or condition is an autoimmune or inflammatorydisease or condition, such as arthritis, e.g., rheumatoid arthritis(RA), Type I diabetes, systemic lupus erythematosus (SLE), inflammatorybowel disease, psoriasis, scleroderma, autoimmune thyroid disease,Grave's disease, Crohn's disease, multiple sclerosis, asthma, and/or adisease or condition associated with transplant.

In some embodiments, the antigen associated with the disease or disorderis selected from the group consisting of orphan tyrosine kinase receptorROR1, B cell maturation antigen (BCMA), carbonic anhydrase 9 (CAIX),tEGFR, Her2/neu (receptor tyrosine kinase erbB2), L1-CAM, CD19, CD20,CD22, mesothelin, CEA, hepatitis B surface antigen, anti-folatereceptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, epithelialglycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrinereceptor A2 (EPHa2), Her2/neu (receptor tyrosine kinase erb-B2), Her3(erb-B3), Her4 (erb-B4), erbB dimers, type III epidermal growth factorreceptor mutation (EGFR vIII), folate binding protein (FBP), FCRL5, Fcreceptor like 5 (FCRL5; also known as Fc receptor homolog 5 or FCRH5),fetal acetylcholine receptor, ganglioside GD2, ganglioside GD3, GProtein Coupled Receptor 5D (GPCR5D), HMW-MAA, IL-22R-alpha,IL-13R-alpha2, kinase insert domain receptor (kdr), kappa light chain,Leucine Rich Repeat Containing 8 Family Member A (LRRC8A), Lewis Y,L1-cell adhesion molecule, (L1-CAM), Melanoma-associated antigen(MAGE)-A1, MAGE-A3, MAGE-A6, Preferentially expressed antigen ofmelanoma (PRAME), survivin, TAG72, B7-H6, IL-13 receptor alpha 2(IL-13Ra2), CA9, GD3, HMW-MAA, CD171, G250/CAIX, Human leukocyte antigenA1 (HLA-A1), MAGE A1, HLA-A2, NY-ESO-1, PSCA, folate receptor-a, CD44v6,CD44v7/8, αvβ6 integrin (avb6 integrin), 8H9, NCAM, VEGF receptors, 5T4,Foetal AchR, natural killer group 2 member D (NKG2D) ligands, CD44v6,dual antigen, a cancer-testes antigen, mesothelin, murine CMV, mucin 1(MUC1), MUC16, prostate stem cell antigen (PSCA), NKG2D, a cancer-testisantigen cancer/testis antigen 1B (CTAG, also known as NY-ESO-1 andLAGE-2), MART-1, glycoprotein 100 (gp100), oncofetal antigen, ROR1,Trophoblast glycoprotein (TPBG also known as 5T4), TAG72, VEGF-R2,carcinoembryonic antigen (CEA), Her2/neu, estrogen receptor,progesterone receptor, ephrinB2, CD123, c-Met, GD-2, O-acetylated GD2(OGD2), CE7, Wilms Tumor 1 (WT-1), a cyclin, cyclin A2, C-C MotifChemokine Ligand 1 (CCL-1), CD138, a pathogen-specific antigen and anantigen associated with a universal tag, and/or biotinylated molecules,and/or molecules expressed by HIV, HCV, HBV or other pathogens.

In some embodiments, the disease or condition is a B cell malignancy. Insome embodiments, the B cell malignancy is a leukemia or a lymphoma. Insome aspects, the disease or condition is acute lymphoblastic leukemia(ALL), adult ALL, chronic lymphoblastic leukemia (CLL), non-Hodgkinlymphoma (NHL), or Diffuse Large B-Cell Lymphoma (DLBCL). In some cases,the disease or condition is an NHL, such as or including an NHL that isan aggressive NHL, diffuse large B cell lymphoma (DLBCL), NOS (de novoand transformed from indolent), primary mediastinal large B celllymphoma (PMBCL), T cell/histocyte-rich large B cell lymphoma (TCHRBCL),Burkitt's lymphoma, mantle cell lymphoma (MCL), and/or follicularlymphoma (FL), optionally, follicular lymphoma Grade 3B (FL3B). In someaspects, the recombinant receptor, such as a CAR, specifically binds toan antigen associated with the disease or condition or expressed incells of the environment of a lesion associated with the B cellmalignancy. Antigens targeted by the receptors in some embodimentsinclude antigens associated with a B cell malignancy, such as any of anumber of known B cell marker. In some embodiments, the antigen targetedby the receptor is CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33,Igkappa, Iglambda, CD79a, CD79b or CD30, or combinations thereof.

In some embodiments, the disease or condition is a myeloma, such as amultiple myeloma. In some aspects, the recombinant receptor, such as aCAR, specifically binds to an antigen associated with the disease orcondition or expressed in cells of the environment of a lesionassociated with the multiple myeloma. Antigens targeted by the receptorsin some embodiments include antigens associated with multiple myeloma.In some aspects, the antigen, e.g., the second or additional antigen,such as the disease-specific antigen and/or related antigen, isexpressed on multiple myeloma, such as B cell maturation antigen (BCMA),G protein-coupled receptor class C group 5 member D (GPRC5D), CD38(cyclic ADP ribose hydrolase), CD138 (syndecan-1, syndecan, SYN-1), CS-1(CS1, CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24), BAFF-R, TACIand/or FcRH5. Other exemplary multiple myeloma antigens include CD56,TIM-3, CD33, CD123, CD44, CD20, CD40, CD74, CD200, EGFR,02-Microglobulin, HM1.24, IGF-1R, IL-6R, TRAIL-R1, and the activinreceptor type IIA (ActRIIA). See Benson and Byrd, J. Clin. Oncol. (2012)30(16): 2013-15; Tao and Anderson, Bone Marrow Research (2011):924058;Chu et al., Leukemia (2013) 28(4):917-27; Garfall et al., Discov Med.(2014) 17(91):37-46. In some embodiments, the antigens include thosepresent on lymphoid tumors, myeloma, AIDS-associated lymphoma, and/orpost-transplant lymphoproliferations, such as CD38. Antibodies orantigen-binding fragments directed against such antigens are known andinclude, for example, those described in U.S. Pat. Nos. 8,153,765;8,603,477, 8,008,450; U.S. Pub. No. US20120189622 or US20100260748;and/or International PCT Publication Nos. WO2006099875, WO2009080829 orWO2012092612 or WO2014210064. In some embodiments, such antibodies orantigen-binding fragments thereof (e.g. scFv) are contained inmultispecific antibodies, multispecific chimeric receptors, such asmultispecific CARs, and/or multispecific cells.

In some embodiments, the antigen is a pathogen-specific orpathogen-expressed antigen. In some embodiments, the antigen is a viralantigen (such as a viral antigen from HIV, HCV, HBV, etc.), bacterialantigens, and/or parasitic antigens.

In some embodiments, the immune cells express a T cell receptor (TCR) orother antigen-binding receptor. In some embodiments, the immune cellsexpress a recombinant receptor, such as a transgenic TCR or a chimericantigen receptor (CAR). In some embodiments, the cells are autologous tothe subject. In some embodiments, the cells are allogeneic to thesubject.

Methods for administration of engineered cells for adoptive cell therapyare known and may be used in connection with the provided methods andcompositions. For example, adoptive T cell therapy methods aredescribed, e.g., in US Patent Application Publication No. 2003/0170238to Gruenberg et al; U.S. Pat. No. 4,690,915 to Rosenberg; Rosenberg(2011) Nat Rev Clin Oncol. 8(10):577-85). See, e.g., Themeli et al.,(2013) Nat Biotechnol. 31(10): 928-933; Tsukahara et al., (2013) BiochemBiophys Res Commun 438(1): 84-9; Davila et al., (2013) PLoS ONE 8(4):e61338.

In some embodiments, the cell therapy, e.g., adoptive T cell therapy, iscarried out by autologous transfer, in which the cells are isolatedand/or otherwise prepared from the subject who is to receive the celltherapy, or from a sample derived from such a subject. Thus, in someaspects, the cells are derived from a subject, e.g., patient, in need ofa treatment and the cells, following isolation and processing areadministered to the same subject.

In some embodiments, the cell therapy, e.g., adoptive T cell therapy, iscarried out by allogeneic transfer, in which the cells are isolatedand/or otherwise prepared from a subject other than a subject who is toreceive or who ultimately receives the cell therapy, e.g., a firstsubject. In such embodiments, the cells then are administered to adifferent subject, e.g., a second subject, of the same species. In someembodiments, the first and second subjects are genetically identical. Insome embodiments, the first and second subjects are genetically similar.In some embodiments, the second subject expresses the same HLA class orsupertype as the first subject.

In certain embodiments, if the risk, probability, and/or likelihood thatthe subject will experience and/or develop a toxicity followingadministration of or associated with the cell therapy is determined tobe high, increased, or elevated by one or more methods as describedherein, then the subject is administered the cell therapy in anin-patient setting and/or with admission to the hospital for one or moredays, optionally wherein the cell therapy is otherwise to beadministered to subjects on an outpatient basis or without admission tothe hospital for one or more days if the risk, probability, and/orlikelihood of is determined to be low, reduced, or decreased accordingto the results of a method as described herein. In particularembodiments, a cell therapy, optionally at a non-reduced dose,optionally on an outpatient basis or without admission to the hospitalfor one or more days, is administered to the subject that is determinedto have a low, decreased, and/or reduced risk of toxicity according tothe results of a method provided herein, such as described in Section I.

In certain embodiments, if the risk, probability, and/or likelihood thatthe subject will experience and/or develop a toxicity followingadministration of or associated with the cell therapy is determined tobe high, increased, or elevated by one or more methods provided inSection I, then the subject is administered a standard dose of the celltherapy in an in-patient setting and/or with admission to the hospitalfor one or more days. In particular embodiments, if the risk,probability, and/or likelihood that the subject will experience and/ordevelop a toxicity following administration of or associated with thecell therapy is determined to be high, increased, or elevated by one ormore methods as described herein, then the subject is administered areduced dose of the cell therapy in an in-patient setting and/or withadmission to the hospital for one or more days. In certain embodiments,if the risk, probability, and/or likelihood that the subject willexperience and/or develop a toxicity following administration of orassociated with the cell therapy is determined to be low, decreased, orreduced by one or more methods as described herein, then the subject isadministered a standard dose of the cell therapy on an outpatient basisor without admission to the hospital for one or more days.

In certain embodiments, if the risk, probability, and/or likelihood thatthe subject will experience and/or develop a toxicity followingadministration of and/or associated with the cell is determined to behigh, increased, or elevated by one or more methods as describedherrein, then the subject is not administered the cell therapy. In suchembodiments, the subject may be administered an alternative therapy,such as a different cell therapy, or an immunotherapy.

In some embodiments, subjects with low, decreased, and/or reduced risk,probability, and/or likelihood to experience and/or develop a toxicityas determined by one or more methods as described herein, areadministered a standard dose of the cell therapy. In some embodiments,subjects with high, increased, or elevated risk, probability, and/orlikelihood to experience and/or develop a toxicity as determined by oneor more methods as described herein, are administered a reduced dose ofthe cell therapy. In some embodiments, subjects with high, increased, orelevated risk, probability, and/or likelihood to experience and/ordevelop a toxicity as determined by one or more methods as describedherein, are administered a standard dose of the cell therapy and anintervention to prevent or reduce toxicity as described herein. Inparticular embodiments, subjects with high, increased, or elevated risk,probability, and/or likelihood to experience and/or develop a toxicityas determined by one or more methods as described herein, areadministered a reduced dose of the cell therapy and an intervention toprevent or reduce toxicity as described herein.

In some embodiments, more than 30%, more than 35%, more than 40%, morethan 45%, more than 50%, more than 55%, more than 60%, more than 65%,more than 70%, more than 85%, more than 90%, more than 97%, more than99%, or about 100% of the subjects do not develop or experience severetoxicity. In some embodiments, the severe toxicity is severe CRS ofgrade 3 or higher, grade 4 or higher, or grade 5. In some embodiments,the severe toxicity is severe neurotoxicity of grade 3 or higher,extended grade 3 or higher, grade 4 or higher, or grade 5.

In some embodiments, at least 30%, at least 35%, at least 40%, at least45%, at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 85%, at least 90%, at least 97%, at least 99%, or about100% of the subjects treated according to the provided methods do notdevelop or experience early toxicity. In some embodiments, earlytoxicity is a toxicity developed in the subject by less than eight,seven, six, five, four or three days after initiation of theadministration of the cell therapy. In some instances, CRS, such assevere CRS, typically occurs 6-20 days after infusion of cells thatexpress a CAR. See Xu et al., Cancer Letters 343 (2014) 172-78. In somecases, the onset of CRS occurs less than or by 4 days afteradministration of the cell therapy.

In certain embodiments, a reduced dose of a therapeutic cell compositioncontains less than or equal to 90%, less than or equal to 80%, less thanor equal to 70%, less than or equal to 60%, less than or equal to 50%,less than or equal to 40%, less than or equal to 30%, less than or equalto 20%, or less than or equal to 10%, or less than or equal to 1%, lessthan or equal to 0.1%, or less than or equal to 0.01% of the totalamount of cells, CAR+ cells, CAR+CD4+ cells, and/or CD8+CAR+ cells of astandard dose of the therapeutic cell composition. In some embodiments,a reduced dose of a therapeutic cell composition contains less than orequal to 90%, less than or equal to 80%, less than or equal to 70%, lessthan or equal to 60%, less than or equal to 50%, less than or equal to40%, less than or equal to 30%, less than or equal to 20%, or less thanor equal to 10%, or less than or equal to 1%, less than or equal to0.1%, or less than or equal to 0.01% of the amount of CAR+ cells of astandard dose of the therapeutic cell composition total amount of cellsper kg of subject body weight, CAR+ cells per kg of subject body weight,CAR+CD4+ cells per kg of subject body weight, and/or CD8+CAR+ cells perkg of subject body weight.

In some embodiments, the methods include administration of theengineered cells or a composition containing the cells to a subject,tissue, or cell, such as one having, at risk for, or suspected of havingthe disease, condition or disorder. In some embodiments, the cells,populations, and compositions are administered to a subject having theparticular disease or condition to be treated, e.g., via adoptive celltherapy, such as adoptive T cell therapy. In some embodiments, the cellsor compositions are administered to the subject, such as a subjecthaving or at risk for the disease or condition, ameliorate one or moresymptom of the disease or condition.

In some embodiments, the cell-based therapy is or comprisesadministration of cells, such as T cells, that target a moleculeexpressed on the surface of a lesion, such as a tumor or a cancer. Insome embodiments, the immune cells express a T cell receptor (TCR) orother antigen-binding receptor. In some embodiments, the immune cellsexpress a recombinant receptor, such as a transgenic TCR or a chimericantigen receptor (CAR). In some embodiments, the cells are autologous tothe subject. In some embodiments, the cells are allogeneic to thesubject.

Methods for administration of engineered cells for adoptive cell therapyare known and may be used in connection with the provided methods andcompositions. For example, adoptive T cell therapy methods aredescribed, e.g., in US Patent Application Publication No. 2003/0170238to Gruenberg et al; U.S. Pat. No. 4,690,915 to Rosenberg; Rosenberg(2011) Nat Rev Clin Oncol. 8(10):577-85). See, e.g., Themeli et al.,(2013) Nat Biotechnol. 31(10): 928-933; Tsukahara et al., (2013) BiochemBiophys Res Commun 438(1): 84-9; Davila et al., (2013) PLoS ONE 8(4):e61338.

In some embodiments, the cell therapy, e.g., adoptive T cell therapy, iscarried out by autologous transfer, in which the cells are isolatedand/or otherwise prepared from the subject who is to receive the celltherapy, or from a sample derived from such a subject. Thus, in someaspects, the cells are derived from a subject, e.g., patient, in need ofa treatment and the cells, following isolation and processing areadministered to the same subject.

In some embodiments, the cell therapy, e.g., adoptive T cell therapy, iscarried out by allogeneic transfer, in which the cells are isolatedand/or otherwise prepared from a subject other than a subject who is toreceive or who ultimately receives the cell therapy, e.g., a firstsubject. In such embodiments, the cells then are administered to adifferent subject, e.g., a second subject, of the same species. In someembodiments, the first and second subjects are genetically identical. Insome embodiments, the first and second subjects are genetically similar.In some embodiments, the second subject expresses the same HLA class orsupertype as the first subject. The cells can be administered by anysuitable means. Dosing and administration may depend in part on whetherthe administration is brief or chronic. Various dosing schedules includebut are not limited to single or multiple administrations over varioustime-points, bolus administration, and pulse infusion.

The cells can be administered by any suitable means, for example, bybolus infusion, by injection, e.g., intravenous or subcutaneousinjections, intraocular injection, periocular injection, subretinalinjection, intravitreal injection, trans-septal injection, subscleralinjection, intrachoroidal injection, intracameral injection,subconjectval injection, subconjuntival injection, sub-Tenon'sinjection, retrobulbar injection, peribulbar injection, or posteriorjuxtascleral delivery. In some embodiments, they are administered byparenteral, intrapulmonary, and intranasal, and, if desired for localtreatment, intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. In some embodiments, a given dose isadministered by a single bolus administration of the cells. In someembodiments, it is administered by multiple bolus administrations of thecells, for example, over a period of no more than 3 days, or bycontinuous infusion administration of the cells. In some embodiments,administration of the cell dose or any additional therapies, e.g., thelymphodepleting therapy, intervention therapy and/or combinationtherapy, is carried out via outpatient delivery.

In some embodiments, the methods comprise administration of achemotherapeutic agent, e.g., a conditioning chemotherapeutic agent, forexample, to reduce tumor burden prior to the administration.

Preconditioning subjects with immunodepleting (e.g., lymphodepleting)therapies in some aspects can improve the effects of adoptive celltherapy (ACT).

Thus, in some embodiments, the methods include administering apreconditioning agent, such as a lymphodepleting or chemotherapeuticagent, such as cyclophosphamide, fludarabine, or combinations thereof,to a subject prior to the initiation of the cell therapy. For example,the subject may be administered a preconditioning agent at least 2 daysprior, such as at least 3, 4, 5, 6, or 7 days prior, to the initiationof the cell therapy. In some embodiments, the subject is administered apreconditioning agent no more than 7 days prior, such as no more than 6,5, 4, 3, or 2 days prior, to the initiation of the cell therapy.

In some embodiments, the subject is preconditioned with cyclophosphamideat a dose between or between about 20 mg/kg and 100 mg/kg, such asbetween or between about 40 mg/kg and 80 mg/kg. In some aspects, thesubject is preconditioned with or with about 60 mg/kg ofcyclophosphamide. In some embodiments, the cyclophosphamide can beadministered in a single dose or can be administered in a plurality ofdoses, such as given daily, every other day or every three days. In someembodiments, the cyclophosphamide is administered once daily for one ortwo days. In some embodiments, where the lymphodepleting agent comprisescyclophosphamide, the subject is administered cyclophosphamide at a dosebetween or between about 100 mg/m² and 500 mg/m², such as between orbetween about 200 mg/m² and 400 mg/m2, or 250 mg/m² and 350 mg/m²,inclusive. In some instances, the subject is administered about 300mg/m² of cyclophosphamide. In some embodiments, the cyclophosphamide canbe administered in a single dose or can be administered in a pluralityof doses, such as given daily, every other day or every three days. Insome embodiments, cyclophosphamide is administered daily, such as for1-5 days, for example, for 3 to 5 days. In some instances, the subjectis administered about 300 mg/m² of cyclophosphamide, daily for 3 days,prior to initiation of the cell therapy.

In some embodiments, where the lymphodepleting agent comprisesfludarabine, the subject is administered fludarabine at a dose betweenor between about 1 mg/m² and 100 mg/m², such as between or between about10 mg/m² and 75 mg/m², 15 mg/m² and 50 mg/m², 20 mg/m² and 40 mg/m², or24 mg/m² and 35 mg/m², inclusive. In some instances, the subject isadministered about 30 mg/m² of fludarabine. In some embodiments, thefludarabine can be administered in a single dose or can be administeredin a plurality of doses, such as given daily, every other day or everythree days. In some embodiments, fludarabine is administered daily, suchas for 1-5 days, for example, for 3 to 5 days. In some instances, thesubject is administered about 30 mg/m² of fludarabine, daily for 3 days,prior to initiation of the cell therapy.

In some embodiments, the lymphodepleting agent comprises a combinationof agents, such as a combination of cyclophosphamide and fludarabine.Thus, the combination of agents may include cyclophosphamide at any doseor administration schedule, such as those described above, andfludarabine at any dose or administration schedule, such as thosedescribed above. For example, in some aspects, the subject isadministered 60 mg/kg (˜2 g/m²) of cyclophosphamide and 3 to 5 doses of25 mg/m² fludarabine prior to the first or subsequent dose.

In some embodiments, the provided methods include one or more steps ofadministering to a subject cells therapeuticcell composition, such as acomposition of cells described herein, e.g. in Section II. In certainembodiments, the cells of the therapeutic cell composition includeengineered CD4+ T cells and engineered CD8+ T cells. In someembodiments, the engineered CD4+ and CD8+ T cells express a T cellreceptor (TCR) or other antigen-binding receptor. In some embodiments,the immune cells express a recombinant receptor, such as a transgenicTCR or a chimeric antigen receptor (CAR). In some embodiments, the cellsof the therapeutic cell composition are autologous to the subject. Insome embodiments, the cells are allogeneic to the subject.

In certain embodiments, the CD4+ T cells and CD8+ T cells of thetherapeutic cell composition are administered to the subject in the samecomposition, dose, or mixture. Thus, in some embodiments, therecombinant receptor expressing CD4+ T cells and recombinant receptorexpressing CD8+ T cells, e.g., CAR+CD4+ and CAR+CD8+ are administered tothe subject in the same composition, dose, or mixture.

2. Dosing

In certain embodiments, if the risk, probability, and/or likelihood thatthe subject will experience and/or develop a toxicity followingadministration of or associated with the cell therapy is determined tobe high, increased, or elevated by one or more of the methods provided,e.g. as described in Section I, then the subject is administered areduced dose of the cell therapy. In certain embodiments, if the risk,probability, and/or likelihood that the subject will experience and/ordevelop a toxicity following administration of or associated with thecell therapy is determined to be low, reduced, or decreased by one ormore of the methods provided, e.g. as described in Section I, then thesubject is administered a standard dose of the cell therapy.

In some embodiments, a reduced dose is less than or equal to 90%, 85%,80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%,10%, 5%, 3%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, 0.001%, or 0.0001% of astandard dose.

In certain embodiments, a reduced dose of a therapeutic cell compositioncontains less than or equal to 90%, less than or equal to 80%, less thanor equal to 70%, less than or equal to 60%, less than or equal to 50%,less than or equal to 40%, less than or equal to 30%, less than or equalto 20%, or less than or equal to 10%, or less than or equal to 1%, lessthan or equal to 0.1%, or less than or equal to 0.01% of the totalamount of cells, CAR+ cells, CAR+CD4+ cells, and/or CD8+CAR+ cells of astandard dose of the therapeutic cell composition. In some embodiments,a reduced dose of a therapeutic cell composition contains less than orequal to 90%, less than or equal to 80%, less than or equal to 70%, lessthan or equal to 60%, less than or equal to 50%, less than or equal to40%, less than or equal to 30%, less than or equal to 20%, or less thanor equal to 10%, or less than or equal to 1%, less than or equal to0.1%, or less than or equal to 0.01% of the amount of CAR+ cells of astandard dose of the therapeutic cell composition total amount of cellsper kg of subject body weight, CAR+ cells per kg of subject body weight,CAR+CD4+ cells per kg of subject body weight, and/or CD8+CAR+ cells perkg of subject body weight.

In some embodiments, the standard dose of cells comprises between at orabout 2×10⁵ of the cells/kg and at or about 2×10⁶ of the cells/kg, suchas between at or about 4×10⁵ of the cells/kg and at or about 1×10⁶ ofthe cells/kg or between at or about 6×10⁵ of the cells/kg and at orabout 8×10⁵ of the cells/kg. In some embodiments, the standard dose ofcells comprises no more than 2×10⁵ of the cells (e.g.antigen-expressing, such as CAR-expressing cells) per kilogram bodyweight of the subject (cells/kg), such as no more than at or about 3×10⁵cells/kg, no more than at or about 4×10⁵ cells/kg, no more than at orabout 5×10⁵ cells/kg, no more than at or about 6×10⁵ cells/kg, no morethan at or about 7×10⁵ cells/kg, no more than at or about 8×10⁵cells/kg, no more than at or about 9×10⁵ cells/kg, no more than at orabout 1×10⁶ cells/kg, or no more than at or about 2×10⁶ cells/kg. Insome embodiments, the standard dose of cells comprises at least or atleast about or at or about 2×10⁵ of the cells (e.g. antigen-expressing,such as CAR-expressing cells) per kilogram body weight of the subject(cells/kg), such as at least or at least about or at or about 3×10⁵cells/kg, at least or at least about or at or about 4×10⁵ cells/kg, atleast or at least about or at or about 5×10⁵ cells/kg, at least or atleast about or at or about 6×10⁵ cells/kg, at least or at least about orat or about 7×10⁵ cells/kg, at least or at least about or at or about8×10⁵ cells/kg, at least or at least about or at or about 9×10⁵cells/kg, at least or at least about or at or about 1×10⁶ cells/kg, orat least or at least about or at or about 2×10⁶ cells/kg.

In certain embodiments, the cells are administered to the subject at astandard dose. In particular embodiments, the standard dose is orcontains a range of about one million to about 100 billion cells and/orthat amount of cells per kilogram of body weight, such as, e.g., 1million to about 50 billion cells (e.g., about 5 million cells, about 25million cells, about 500 million cells, about 1 billion cells, about 5billion cells, about 20 billion cells, about 30 billion cells, about 40billion cells, or a range defined by any two of the foregoing values),such as about 10 million to about 100 billion cells (e.g., about 20million cells, about 30 million cells, about 40 million cells, about 60million cells, about 70 million cells, about 80 million cells, about 90million cells, about 10 billion cells, about 25 billion cells, about 50billion cells, about 75 billion cells, about 90 billion cells, or arange defined by any two of the foregoing values), and in some casesabout 100 million cells to about 50 billion cells (e.g., about 120million cells, about 250 million cells, about 350 million cells, about450 million cells, about 650 million cells, about 800 million cells,about 900 million cells, about 3 billion cells, about 30 billion cells,about 45 billion cells) or any value in between these ranges and/or perkilogram of body weight. Dosages may vary depending on attributesparticular to the disease or disorder and/or patient and/or othertreatments.

In some embodiments, for example, where the subject is a human, the doseincludes fewer than about 5×10⁸ total recombinant receptor (e.g.,CAR)-expressing cells, T cells, or peripheral blood mononuclear cells(PBMCs). In some embodiments, for example, where the subject is a human,the standard dose includes fewer than about 1×10⁸ total recombinantreceptor (e.g., CAR)-expressing cells, T cells, or peripheral bloodmononuclear cells (PBMCs), e.g., in the range of about 1×10⁶ to 1×10⁸such cells, such as 2×10⁶, 5×10⁶, 1×10⁷, 5×10⁷, or 1×10⁸ or total suchcells, or the range between any two of the foregoing values.

In some embodiments, the dose of genetically engineered cells comprisesfrom or from about 1×10⁵ to 5×10⁸ total CAR-expressing T cells, 1×10⁵ to2.5×10⁸ total CAR-expressing T cells, 1×10⁵ to 1×10⁸ totalCAR-expressing T cells, 1×10⁵ to 5×10⁷ total CAR-expressing T cells,1×10⁵ to 2.5×10⁷ total CAR-expressing T cells, 1×10⁵ to 1×10⁷ totalCAR-expressing T cells, 1×10⁵ to 5×10⁶ total CAR-expressing T cells,1×10⁵ to 2.5×10⁶ total CAR-expressing T cells, 1×10⁵ to 1×10⁶ totalCAR-expressing T cells, 1×10⁶ to 5×10⁸ total CAR-expressing T cells,1×10⁶ to 2.5×10⁸ total CAR-expressing T cells, 1×10⁶ to 1×10⁸ totalCAR-expressing T cells, 1×10⁶ to 5×10⁷ total CAR-expressing T cells,1×10⁶ to 2.5×10⁷ total CAR-expressing T cells, 1×10⁶ to 1×10⁷ totalCAR-expressing T cells, 1×10⁶ to 5×10⁶ total CAR-expressing T cells,1×10⁶ to 2.5×10⁶ total CAR-expressing T cells, 2.5×10⁶ to 5×10⁸ totalCAR-expressing T cells, 2.5×10⁶ to 2.5×10⁸ total CAR-expressing T cells,2.5×10⁶ to 1×10⁸ total CAR-expressing T cells, 2.5×10⁶ to 5×10⁷ totalCAR-expressing T cells, 2.5×10⁶ to 2.5×10⁷ total CAR-expressing T cells,2.5×10⁶ to 1×10⁷ total CAR-expressing T cells, 2.5×10⁶ to 5×10⁶ totalCAR-expressing T cells, 5×10⁶ to 5×10⁸ total CAR-expressing T cells,5×10⁶ to 2.5×10⁸ total CAR-expressing T cells, 5×10⁶ to 1×10⁸ totalCAR-expressing T cells, 5×10⁶ to 5×10⁷ total CAR-expressing T cells,5×10⁶ to 2.5×10⁷ total CAR-expressing T cells, 5×10⁶ to 1×10⁷ totalCAR-expressing T cells, 1×10⁷ to 5×10⁸ total CAR-expressing T cells,1×10⁷ to 2.5×10⁸ total CAR-expressing T cells, 1×10⁷ to 1×10⁸ totalCAR-expressing T cells, 1×10⁷ to 5×10⁷ total CAR-expressing T cells,1×10⁷ to 2.5×10⁷ total CAR-expressing T cells, 2.5×10⁷ to 5×10⁸ totalCAR-expressing T cells, 2.5×10⁷ to 2.5×10⁸ total CAR-expressing T cells,2.5×10⁷ to 1×10⁸ total CAR-expressing T cells, 2.5×10⁷ to 5×10⁷ totalCAR-expressing T cells, 5×10⁷ to 5×10⁸ total CAR-expressing T cells,5×10⁷ to 2.5×10⁸ total CAR-expressing T cells, 5×10⁷ to 1×10⁸ totalCAR-expressing T cells, 1×10⁸ to 5×10⁸ total CAR-expressing T cells,1×10⁸ to 2.5×10⁸ total CAR-expressing T cells, or 2.5×10⁸ totalCAR-expressing T cells.

In some embodiments, the dose of genetically engineered cells comprisesat least or at least about 1×10⁵ CAR-expressing cells, at least or atleast about 2.5×10⁵ CAR-expressing cells, at least or at least about5×10⁵ CAR-expressing cells, at least or at least about 1×10⁶CAR-expressing cells, at least or at least about 2.5×10⁶ CAR-expressingcells, at least or at least about 5×10⁶ CAR-expressing cells, at leastor at least about 1×10⁷ CAR-expressing cells, at least or at least about2.5×10⁷ CAR-expressing cells, at least or at least about 5×10⁷CAR-expressing cells, at least or at least about 1×10⁸ CAR-expressingcells, at least or at least about 2.5×10⁸ CAR-expressing cells, or atleast or at least about 5×10⁸ CAR-expressing cells.

In some embodiments, the cell therapy comprises administration of a dosecomprising a number of cell from or from about 1×10⁵ to 5×10⁸ totalrecombinant receptor-expressing cells, total T cells, or totalperipheral blood mononuclear cells (PBMCs), from or from about 5×10⁵ to1×10⁷ total recombinant receptor-expressing cells, total T cells, ortotal peripheral blood mononuclear cells (PBMCs) or from or from about1×10⁶ to 1×10⁷ total recombinant receptor-expressing cells, total Tcells, or total peripheral blood mononuclear cells (PBMCs), eachinclusive. In some embodiments, the cell therapy comprisesadministration of a dose of cells comprising a number of cells at leastor at least about 1×10⁵ total recombinant receptor-expressing cells,total T cells, or total peripheral blood mononuclear cells (PBMCs), suchat least or at least 1×10⁶, at least or at least about 1×10⁷, at leastor at least about 1×10⁸ of such cells. In some embodiments, the numberis with reference to the total number of CD3+ or CD8+, in some casesalso recombinant receptor-expressing (e.g. CAR+) cells. In someembodiments, the cell therapy comprises administration of a dosecomprising a number of cell from or from about 1×10⁵ to 5×10⁸ CD3+ orCD8+ total T cells or CD3+ or CD8+ recombinant receptor-expressingcells, from or from about 5×10⁵ to 1×10⁷ CD3+ or CD8+ total T cells orCD3+ or CD8+ recombinant receptor-expressing cells, or from or fromabout 1×10⁶ to 1×10⁷ CD3+ or CD8+ total T cells or CD3+ or CD8+recombinant receptor-expressing cells, each inclusive. In someembodiments, the cell therapy comprises administration of a dosecomprising a number of cell from or from about 1×10⁵ to 5×10⁸ totalCD3+/CAR+ or CD8+/CAR+ cells, from or from about 5×10⁵ to 1×10⁷ totalCD3+/CAR+ or CD8+/CAR+ cells, or from or from about 1×10⁶ to 1×10⁷ totalCD3+/CAR+ or CD8+/CAR+ cells, each inclusive.

In some embodiments, the T cells of the dose include CD4+ T cells, CD8+T cells or CD4+ and CD8+ T cells.

In some embodiments, for example, where the subject is human, the CD8+ Tcells of the dose, including in a dose including CD4+ and CD8+ T cells,includes between about 1×10⁶ and 5×10⁸ total recombinant receptor (e.g.,CAR)-expressing CD8+ cells, e.g., in the range of about 5×10⁶ to 1×10⁸such cells, such cells 1×10⁷, 2.5×10⁷, 5×10⁷, 7.5×10⁷, 1×10⁸, or 5×10⁸total such cells, or the range between any two of the foregoing values.In some embodiments, the patient is administered multiple doses, andeach of the doses or the total dose can be within any of the foregoingvalues. In some embodiments, the dose of cells comprises theadministration of from or from about 1×10⁷ to 0.75×10⁸ total recombinantreceptor-expressing CD8+ T cells, 1×10⁷ to 2.5×10⁷ total recombinantreceptor-expressing CD8+ T cells, from or from about 1×10⁷ to 0.75×10⁸total recombinant receptor-expressing CD8+ T cells, each inclusive. Insome embodiments, the dose of cells comprises the administration of orabout 1×10⁷, 2.5×10⁷, 5×10⁷ 7.5×10⁷, 1×10⁸, or 5×10⁸ total recombinantreceptor-expressing CD8+ T cells.

In some embodiments, the dose of cells, e.g., recombinantreceptor-expressing T cells, is administered to the subject as a singledose or is administered only one time within a period of two weeks, onemonth, three months, six months, 1 year or more.

In some aspects, the pharmaceutical compositions and formulations areprovided as unit dose form compositions including the number of cellsfor administration in a given dose or fraction thereof. In someembodiments, the provided methods produce cells in a predictabletimeline to dosing as compared to other methods of incubating (e.g.,stimulating) cells. In some cases, the dose of cells for administrationis determined based on the number of naïve-like cells in the input cellcomposition. In some embodiments, the unit dose is a standard dose.

In some aspects, the size of a dose, e.g., standard dose is determinedby the burden of the disease or condition in the subject. For example,in some aspects, the number of cells administered in the dose isdetermined based on the tumor burden that is present in the subjectimmediately prior to administration of the initiation of the dose ofcells. In some embodiments, the size of the first and/or subsequentstandard dose is inversely correlated with disease burden. In someaspects, as in the context of a large disease burden, the subject isadministered a low number of cells. In other embodiments, as in thecontext of a lower disease burden, the subject is administered a largernumber of cells.

In some aspects, the size of the standard dose is determined by thesubject's risk for toxicity, e.g., as assessed by the fold change in afactor indicative of disease burden. In some aspects, the number ofcells administered in the dose is determined based on the fold change ina factor indicative of disease burden between a first time point and asecond time point prior to administration of the therapy. In someembodiments, the size of the first and/or subsequent dose is inverselycorrelated with the risk for toxicity. In some aspects, as in thecontext of a likelihood and/or elevated risk or probability of toxicity,the subject is administered a low number of cells. In other embodiments,as in the context of a lower risk or probability, the subject isadministered a larger number of cells.

In some aspects, the size of the standard dose is determined by theactivity of the cells of the cell therapy, e.g., as determined bymeasuring one or more parameters of the cell therapy. In some aspects,the number of cells administered in the dose is determined based on theactivity of the cells of the cell therapy. In some embodiments, the sizeof the first and/or subsequent dose is inversely correlated with theamount or level of activity in the cells. In particular embodiments, thenumber of cells administered in the dose is determined by the activityof the cells of the cell therapy and the risk of the subject for atoxicity. In certain embodiments, the subject is administered a standarddose of the cell therapy if the cells have a low or moderate activity asdetermined by measuring the one or more parameters, regardless of therisk assessment provided by the fold change in a factor indicative ofdisease burden in the subject.

Following administration of the cells, the biological activity of theengineered cell populations in some embodiments is measured, e.g., byany of a number of known methods. Parameters to assess include specificbinding of an engineered or natural T cell or other immune cell toantigen, in vivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flowcytometry. In certain embodiments, the ability of the engineered cellsto destroy target cells can be measured using any suitable method, suchas cytotoxicity assays described in, for example, Kochenderfer et al.,J. Immunotherapy, 32(7): 689-702 (2009), and Herman et al. J.Immunological Methods, 285(1): 25-40 (2004). In certain embodiments, thebiological activity of the cells is measured by assaying expressionand/or secretion of one or more cytokines, such as CD 107a, IFNγ, IL-2,and TNF. In some aspects the biological activity is measured byassessing clinical outcome, such as reduction in tumor burden or load.

In some embodiments, cells of the dose may be administered byadministration of a plurality of compositions or solutions, such as afirst and a second, optionally more, each containing some cells of thedose. In some aspects, the plurality of compositions, each containing adifferent population and/or sub-types of cells, are administeredseparately or independently, optionally within a certain period of time.For example, the populations or sub-types of cells can include CD8⁺ andCD4⁺ T cells, respectively, and/or CD8+- and CD4+-enriched populations,respectively, e.g., CD4+ and/or CD8+ T cells each individually includingcells genetically engineered to express the recombinant receptor. Insome embodiments, the administration of the dose comprisesadministration of a first composition comprising a dose of CD8+ T cellsor a dose of CD4+ T cells and administration of a second compositioncomprising the other of the dose of CD4+ T cells and the CD8+ T cells.

In some embodiments, the administration of the composition or dose,e.g., administration of the plurality of cell compositions, involvesadministration of the cell compositions separately. In some aspects, theseparate administrations are carried out simultaneously, orsequentially, in any order. In some embodiments, the dose comprises afirst composition and a second composition, and the first compositionand second composition are administered 0 to 12 hours apart, 0 to 6hours apart or 0 to 2 hours apart. In some embodiments, the initiationof administration of the first composition and the initiation ofadministration of the second composition are carried out no more than 2hours, no more than 1 hour, or no more than 30 minutes apart, no morethan 15 minutes, no more than 10 minutes or no more than 5 minutesapart. In some embodiments, the initiation and/or completion ofadministration of the first composition and the completion and/orinitiation of administration of the second composition are carried outno more than 2 hours, no more than 1 hour, or no more than 30 minutesapart, no more than 15 minutes, no more than 10 minutes or no more than5 minutes apart.

In some composition, the first composition, e.g., first composition ofthe dose, comprises CD4+ T cells. In some composition, the firstcomposition, e.g., first composition of the dose, comprises CD8+ Tcells. In some embodiments, the first composition is administered priorto the second composition.

In some embodiments, the dose or composition of cells includes a definedor target ratio of CD4+ cells expressing a recombinant receptor to CD8+cells expressing a recombinant receptor and/or of CD4+ cells to CD8+cells, which ratio optionally is approximately 1:1 or is betweenapproximately 1:3 and approximately 3:1, such as approximately 1:1. Insome aspects, the administration of a composition or dose with thetarget or desired ratio of different cell populations (such as CD4+:CD8+ratio or CAR+CD4+:CAR+CD8+ ratio, e.g., 1:1) involves the administrationof a cell composition containing one of the populations and thenadministration of a separate cell composition comprising the other ofthe populations, where the administration is at or approximately at thetarget or desired ratio. In some aspects, administration of a dose orcomposition of cells at a defined ratio leads to improved expansion,persistence and/or antitumor activity of the T cell therapy.

In some embodiments, the subject receives multiple doses, e.g., two ormore doses or multiple consecutive doses, of the cells. In someembodiments, two doses are administered to a subject. In someembodiments, the subject receives the consecutive dose, e.g., seconddose, is administered approximately 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20 or 21 days after the first dose. In someembodiments, multiple consecutive doses are administered following thefirst dose, such that an additional dose or doses are administeredfollowing administration of the consecutive dose. In some aspects, thenumber of cells administered to the subject in the additional dose isthe same as or similar to the first dose and/or consecutive dose. Insome embodiments, the additional dose or doses are larger than priordoses.

In some aspects, the size of the first and/or consecutive dose isdetermined based on one or more criteria such as response of the subjectto prior treatment, e.g. chemotherapy, disease burden in the subject,such as tumor load, bulk, size, or degree, extent, or type ofmetastasis, stage, and/or likelihood or incidence of the subjectdeveloping toxic outcomes, e.g., CRS, macrophage activation syndrome,tumor lysis syndrome, neurotoxicity, and/or a host immune responseagainst the cells and/or recombinant receptors being administered.

In some aspects, the time between the administration of the first doseand the administration of the consecutive dose is about 9 to about 35days, about 14 to about 28 days, or 15 to 27 days. In some embodiments,the administration of the consecutive dose is at a time point more thanabout 14 days after and less than about 28 days after the administrationof the first dose. In some aspects, the time between the first andconsecutive dose is about 21 days. In some embodiments, an additionaldose or doses, e.g. consecutive doses, are administered followingadministration of the consecutive dose. In some aspects, the additionalconsecutive dose or doses are administered at least about 14 and lessthan about 28 days following administration of a prior dose. In someembodiments, the additional dose is administered less than about 14 daysfollowing the prior dose, for example, 4, 5, 6, 7, 8, 9, 10, 11, 12, or13 days after the prior dose. In some embodiments, no dose isadministered less than about 14 days following the prior dose and/or nodose is administered more than about 28 days after the prior dose.

In some embodiments, the dose of cells, e.g., recombinantreceptor-expressing cells, comprises two doses (e.g., a double dose),comprising a first dose of the T cells and a consecutive dose of the Tcells, wherein one or both of the first dose and the second dosecomprises administration of the split dose of T cells.

In some embodiments, the cells are administered as part of a furthercombination treatment, such as simultaneously with or sequentially with,in any order, another therapeutic intervention, such as an antibody orengineered cell or receptor or agent, such as a cytotoxic or therapeuticagent. For example, in some embodiments, an anti-cancer agent orimmunomodulatory agent can be used in combination therapy with adoptivecell therapy with engineered cell expressing a recombinant receptor,e.g. a CAR. In some contexts, the cells are co-administered with anothertherapy sufficiently close in time such that the cell populationsenhance the effect of the P one or more additional therapeutic agents,or vice versa. In some embodiments, the cells are administered prior tothe one or more additional therapeutic agents. In some embodiments, thecells are administered after the one or more additional therapeuticagents.

In some embodiments, the one or more additional therapeutic agentsinclude a cytokine, such as IL-2, for example, to enhance persistence.In some embodiments, the methods comprise administration of achemotherapeutic agent. In some embodiments, the one or more additionaltherapeutic agents include one or more lymphodepleting therapies, suchas prior to or simultaneous with initiation of administration of theengineered cells. In some embodiments, the lymphodepleting therapycomprises administration of a phosphamide, such as cyclophosphamide. Insome embodiments, the lymphodepleting therapy can include administrationof fludarabine. In some embodiments, fludarabine is excluded in thelymphodepleting therapy. In some embodiments, a lymphodepleting therapyis not administered.

In some embodiments, the methods include administering a preconditioningagent, such as a lymphodepleting or chemotherapeutic agent, such ascyclophosphamide, fludarabine, or combinations thereof, to a subjectprior to the initiation of the cell therapy. For example, the subjectmay be administered a preconditioning agent at least 2 days prior, suchas at least 3, 4, 5, 6, or 7 days prior, to the initiation of the celltherapy. In some embodiments, the subject is administered apreconditioning agent no more than 7 days prior, such as no more than 6,5, 4, 3, or 2 days prior, to the initiation of the cell therapy.

In some embodiments, the subject is preconditioned with cyclophosphamideat a dose between or between about 20 mg/kg and 100 mg/kg, such asbetween or between about 40 mg/kg and 80 mg/kg. In some aspects, thesubject is preconditioned with or with about 60 mg/kg ofcyclophosphamide. In some embodiments, the cyclophosphamide can beadministered in a single dose or can be administered in a plurality ofdoses, such as given daily, every other day or every three days. In someembodiments, the cyclophosphamide is administered once daily for one ortwo days. In some embodiments, where the lymphodepleting agent comprisescyclophosphamide, the subject is administered cyclophosphamide at a dosebetween or between about 100 mg/m² and 500 mg/m², such as between orbetween about 200 mg/m² and 400 mg/m² or 250 mg/m² and 350 mg/m²,inclusive. In some instances, the subject is administered about 300mg/m² of cyclophosphamide. In some embodiments, the cyclophosphamide canbe administered in a single dose or can be administered in a pluralityof doses, such as given daily, every other day or every three days. Insome embodiments, cyclophosphamide is administered daily, such as for1-5 days, for example, for 3 to 5 days. In some instances, the subjectis administered about 300 mg/m² of cyclophosphamide, daily for 3 days,prior to initiation of the cell therapy.

In some embodiments, where the lymphodepleting agent comprisesfludarabine, the subject is administered fludarabine at a dose betweenor between about 1 mg/m² and 100 mg/m², such as between or between about10 mg/m² and 75 mg/m², 15 mg/m² and 50 mg/m², 20 mg/m² and 40 mg/m², or24 mg/m² and 35 mg/m², inclusive. In some instances, the subject isadministered about 30 mg/m² of fludarabine. In some embodiments, thefludarabine can be administered in a single dose or can be administeredin a plurality of doses, such as given daily, every other day or everythree days. In some embodiments, fludarabine is administered daily, suchas for 1-5 days, for example, for 3 to 5 days. In some instances, thesubject is administered about 30 mg/m² of fludarabine, daily for 3 days,prior to initiation of the cell therapy.

In some embodiments, the lymphodepleting agent comprises a combinationof agents, such as a combination of cyclophosphamide and fludarabine.Thus, the combination of agents may include cyclophosphamide at any doseor administration schedule, such as those described above, andfludarabine at any dose or administration schedule, such as thosedescribed above. For example, in some aspects, the subject isadministered 60 mg/kg (˜2 g/m²) of cyclophosphamide and 3 to 5 doses of25 mg/m² fludarabine prior to the first or subsequent dose.

The cells can be administered by any suitable means. The cells areadministered in a dosing regimen to achieve a therapeutic effect, suchas a reduction in tumor burden. Dosing and administration may depend inpart on the schedule of administration of the immunomodulatory compound,which can be administered prior to, subsequent to and/or simultaneouslywith initiation of administration of the T cell therapy. Various dosingschedules of the T cell therapy include but are not limited to single ormultiple administrations over various time-points, bolus administration,and pulse infusion. In certain embodiments, the engineered T cellsexpress a recombinant receptor. In certain embodiments, the engineered Tcells express a CAR.

In particular embodiments, the ratio of the CD4+ T cells to CD8+ T cellsthat are administered to the subject in the same composition, dose, ormixture is between 5:1 to 0.2:1, between 4:1 to 0.25:1, between 3:1 to0.33:1, between 2:1 to 0.5:1, between 1.5:1 to 0.66:1, or between 1.25:1to 0.8:1. In some embodiments, the ratio of CD4+ T cells to CD8+ T cellsadministered to the subject in the same composition, dose, or mixture isor is about 2.0:1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1,1.2:1, 1.1:1, 1.0:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, or 0.5:1.

In particular embodiments, the ratio of recombinant receptor expressingCD4+ T cells to recombinant receptor expressing CD8+ T cells that areadministered to the subject in the same composition, dose, or mixture isbetween 5:1 to 0.2:1, between 4:1 to 0.25:1, between 3:1 to 0.33:1,between 2:1 to 0.5:1, between 1.5:1 to 0.66:1, or between 1.25:1 to0.8:1. In certain embodiments, the ratio of recombinant receptorexpressing CD4+ T cells to recombinant receptor expressing CD8+ T cellsthat are administered to the subject in the same composition, dose, ormixture is or is about 2.0:1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1,1.3:1, 1.2:1, 1.1:1, 1.0:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, or 0.5:1. Inparticular embodiments, ratio of the administered recombinant receptorexpressing CD4+ T cells to recombinant receptor expressing CD8+ T cellsis or is about 1:1. In some embodiments, the recombinant receptor is aTCR or a CAR. In particular embodiments, the recombinant receptor is aCAR.

In some embodiments, the ratio of engineered CD4+ T cells to engineeredCD8+ T cells of the dose, composition, or mixture that is administeredto the subject is within a certain tolerated difference or range oferror of such a defined, desired, or fixed ratio. In some embodiments,the tolerated difference is within of or of about 1%, 2%, 3%, 4%, 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%, of the target, defined,preferred, and/or fixed ratio.

In some embodiments, a composition of cells produced by the methodsprovided herein, e.g., an output composition, having a ratio ofengineered CD4+ T cells to engineered CD8+ T cells of between 2:1 and0.5:1 is administered to a subject in a single composition, dose, ormixture. In certain embodiments, the composition contains a ratio ofengineered CD4+ T cells to engineered CD8+ T cells of or of about 1:1.In some embodiments, a cell composition produced from an input cellcomposition, e.g., an input cell composition described in Section I-A1,has a ratio of engineered CD4+ T cells to engineered CD8+ T cells ofbetween 2:1 and 0.5:1 and is administered to a subject in a singlecomposition, dose, or mixture. In particular embodiments, the cellcomposition produced from an input cell composition has a ratio ofengineered CD4+ T cells to engineered CD8+ T cells of 1:1 with atolerated difference of 50%, 25%, 10%, or less.

III. TOXICITY AND INTERVENTIONS OR AGENTS THAT TREAT OR AMELIORATESYMPTOMS OF TOXICITY

In some aspects, the provided embodiments are based on observations thatthe certain pre-treatment factors can be used to assess and monitor arisk of a subject developing a toxicity to a therapy, such as a celltherapy, e.g. CAR-T cells. In some aspects, the methods can improveefficacy of a therapy, e.g., a cell therapy, which otherwise may belimited by the development of toxicity, in some cases severe toxicity,in the subject to whom such cells are administered. For example, in somecases, administering a dose of cells expressing a recombinant receptor,e.g. a CAR, can result in toxicity or risk thereof, such as CRS orneurotoxicity. In some cases, while a higher dose of such cells canincrease the efficacy of the treatment, for example, by increasingexposure to the cells such as by promoting expansion and/or persistence,they may also result in an even greater risk of developing a toxicity ora more severe toxicity. The provided methods provide embodiments tomitigate risks of toxicity, and thereby improving efficacy of thetherapy.

In some embodiments, the provided embodiments are designed to or includefeatures that result in a lower degree of risk of toxicity, a toxicoutcome or symptom, toxicity-promoting profile, factor or property, suchas a symptom or outcome associated with or indicative of cytokinerelease syndrome (CRS) or neurotoxicity. In some aspects, the lowerdegree of such risks is compared to other methods in which the subjectand/or a biological sample from the subject has not been assessed for afactor indicative of tumor burden as described and/or in which thetherapy has not been administered to a subject in accord with anassessment of any preexisting risk of the subject to toxicity followingadministration of the therapeutic cell composition, such as based onbased on such factors indicative of tumor burden.

A. Toxicity

In some aspects, the toxic outcome of a therapy, such as a cell therapy,is or is associated with or indicative of cytokine release syndrome(CRS) or severe CRS (sCRS). CRS, e.g., sCRS, can occur in some casesfollowing adoptive T cell therapy and administration to subjects ofother biological products. See Davila et al., Sci Transl Med 6, 224ra25(2014); Brentjens et al., Sci. Transl. Med. 5, 177ra38 (2013); Grupp etal., N. Engl. J. Med. 368, 1509-1518 (2013); and Kochenderfer et al.,Blood 119, 2709-2720 (2012); Xu et al., Cancer Letters 343 (2014)172-78.

Typically, CRS is caused by an exaggerated systemic immune responsemediated by, for example, T cells, B cells, NK cells, monocytes, and/ormacrophages. Such cells may release a large amount of inflammatorymediators such as cytokines and chemokines. Cytokines may trigger anacute inflammatory response and/or induce endothelial organ damage,which may result in microvascular leakage, heart failure, or death.Severe, life-threatening CRS can lead to pulmonary infiltration and lunginjury, renal failure, or disseminated intravascular coagulation. Othersevere, life-threatening toxicities can include cardiac toxicity,respiratory distress, neurologic toxicity and/or hepatic failure.

Outcomes, signs and symptoms of CRS are known and include thosedescribed herein. In some embodiments, where a particular dosage regimenor administration effects or does not effect a given CRS-associatedoutcome, sign, or symptom, particular outcomes, signs, and symptomsand/or quantities or degrees thereof may be specified.

In the context of administering CAR-expressing cells, CRS, such assevere CRS, typically occurs 6-20 days after infusion of cells thatexpress a CAR. See Xu et al., Cancer Letters 343 (2014) 172-78. In somecases, CRS occurs less than 6 days or more than 20 days after CAR T cellinfusion. The incidence and timing of CRS may be related to baselinecytokine levels or tumor burden at the time of infusion. Commonly, CRSinvolves elevated serum levels of interferon (IFN)-γ, tumor necrosisfactor (TNF)-α, and/or interleukin (IL)-2. Other cytokines that may berapidly induced in CRS are IL-1β, IL-6, IL-8, and IL-10.

CRS criteria that appear to correlate with the onset of CRS to predictwhich patients are more likely to be at risk for developing sCRS havebeen developed (see Davilla et al. Science translational medicine. 2014;6(224):224ra25). Factors include fevers, hypoxia, hypotension,neurologic changes, elevated serum levels of inflammatory cytokines,such as a set of seven cytokines (IFNγ, IL-5, IL-6, IL-10, Flt-3L,fractalkine, and GM-CSF) whose treatment-induced elevation can correlatewell with both pretreatment tumor burden and sCRS symptoms. Otherguidelines on the diagnosis and management of CRS are known (see e.g.,Lee et al, Blood. 2014; 124(2):188-95). In some embodiments, thecriteria reflective of CRS grade are those detailed in Table 1B below.

TABLE 1B Exemplary Grading Criteria for CRS Grade Description ofSymptoms 1 Mild Not life-threatening, require only symptomatic treatmentsuch as antipyretics and anti-emetics (e.g., fever, nausea, fatigue,headache, myalgias, malaise) 2 Moderate Require and respond to moderateintervention: Oxygen requirement <40%, or Hypotension responsive tofluids or low dose of a single vasopressor, or Grade 2 organ toxicity(by CTCAE v4.0) 3 Severe Require and respond to aggressive intervention:Oxygen requirement ≥40%, or Hypotension requiring high dose of a singlevasopressor (e.g., norepinephrine ≥20 μg/kg/min, dopamine ≥10 μg/kg/min,phenylephrine ≥200 μg/kg/min, or epinephrine ≥10 μg/kg/min), orHypotension requiring multiple vasopressors (e.g., vasopressin + one ofthe above agents, or combination vasopressors equivalent to ≥20μg/kg/min norepinephrine), or Grade 3 organ toxicity or Grade 4transaminitis (by CTCAE v4.0) 4 Life- Life-threatening: threateningRequirement for ventilator support, or Grade 4 organ toxicity (excludingtransaminitis) 5 Fatal Death

In some embodiments, a subject is deemed to develop “severe CRS”(“sCRS”) in response to or secondary to administration of a cell therapyor dose of cells thereof, if, following administration, the subjectdisplays: (1) fever of at least 38 degrees Celsius for at least threedays; (2) cytokine elevation that includes either (a) a max fold changeof at least 75 for at least two of the following group of sevencytokines compared to the level immediately following theadministration: interferon gamma (IFNγ), GM-CSF, IL-6, IL-10, Flt-3L,fracktalkine, and IL-5 and/or (b) a max fold change of at least 250 forat least one of the following group of seven cytokines compared to thelevel immediately following the administration: interferon gamma (IFN),GM-CSF, IL-6, IL-10, Flt-3L, fracktalkine, and IL-5; and (c) at leastone clinical sign of toxicity such as hypotension (requiring at leastone intravenous vasoactive pressor) or hypoxia (PO₂<90%) or one or moreneurologic disorder(s) (including mental status changes, obtundation,and/or seizures). In some embodiments, severe CRS includes CRS with agrade of 3 or greater, such as set forth in Table 1B.

In some embodiments, outcomes associated with severe CRS or grade 3 CRSor greater, such as grade 4 or greater, such as set forth in Table 1B.In some embodiments, these include one or more of: persistent fever,e.g., fever of a specified temperature, e.g., greater than at or about38 degrees Celsius, for two or more, e.g., three or more, e.g., four ormore days or for at least three consecutive days; fever greater than ator about 38 degrees Celsius; elevation of cytokines, such as a max foldchange, e.g., of at least at or about 75, compared to pre-treatmentlevels of at least two cytokines (e.g., at least two of the groupconsisting of interferon gamma (IFNγ), GM-CSF, IL-6, IL-10, Flt-3L,fracktalkine, and IL-5, and/or tumor necrosis factor alpha (TNFα)), or amax fold change, e.g., of at least at or about 250 of at least one ofsuch cytokines; and/or at least one clinical sign of toxicity, such ashypotension (e.g., as measured by at least one intravenous vasoactivepressor); hypoxia (e.g., plasma oxygen (PO₂) levels of less than at orabout 90%); and/or one or more neurologic disorders (including mentalstatus changes, obtundation, and seizures). In some embodiments, severeCRS includes CRS that requires management or care in the intensive careunit (ICU).

In some embodiments, severe CRS encompasses a combination of (1)persistent fever (fever of at least 38 degrees Celsius for at leastthree days) and (2) a serum level of CRP of at least at or about 20mg/dL. In some embodiments, severe CRS encompasses hypotension requiringthe use of two or more vasopressors or respiratory failure requiringmechanical ventilation. In some embodiments, the dosage of vasopressorsis increased in a second or subsequent administration.

In some embodiments, the toxicity is evident by the presence of a feverin the subject following administration of a therapy, such as celltherapy (e.g. CAR-T cells). In some embodiments, the fever in thesubject is characterized as a body temperature of the subject that is(or is measured at) at or above a certain threshold temperature orlevel. In some aspects, the threshold temperature is that associatedwith at least a low-grade fever, with at least a moderate fever, and/orwith at least a high-grade fever. In some embodiments, the thresholdtemperature is a particular temperature or range. For example, thethreshold temperature may be at or about 38, 39, 40, 41, or 42 degreesCelsius, and/or may be a range of at or about 38 degrees Celsius to ator about 39 degrees Celsius, a range of at or about 39 degrees Celsiusto at or about 40 degrees Celsius, a range of at or about 40 degreesCelsius to at or about 41 degrees, or a range of at or about 41 degreesCelsius to at or about 42 degrees Celsius.

In some embodiments, the toxicity is associated with a sustained fever,following administration of the therapy, such as cell therapy, e.g.CAR-T cell therapy. In some embodiments, the subject has, and/or isdetermined to or considered to have, a sustained fever if he or sheexhibits a fever at or above the relevant threshold temperature, andwhere the fever or body temperature of the subject does not fluctuate byabout, or by more than about, 1° C., and generally does not fluctuate byabout, or by more than about, 0.5° C., 0.4° C., 0.3° C., or 0.2° C. Suchabsence of fluctuation above or at a certain amount generally ismeasured over a given period of time (such as over a 24-hour, 12-hour,8-hour, 6-hour, 3-hour, or 1-hour period of time, which may be measuredfrom the first sign of fever or the first temperature above theindicated threshold). For example, in some embodiments, a subject isconsidered to or is determined to exhibit sustained fever if he or sheexhibits a fever of at least at or about 38 or 39 degrees Celsius, whichdoes not fluctuate in temperature by more than at or about 0.5° C., 0.4°C., 0.3° C., or 0.2° C., over a period of 6 hours, over a period of 8hours, or over a period of 12 hours, or over a period of 24 hours.

In some embodiments, the subject has, and/or is determined to orconsidered to have, a sustained fever if he or she exhibits a fever ator above the relevant threshold temperature, and where the fever or bodytemperature of the subject is not reduced, or is not reduced by or bymore than a specified amount (e.g., by more than 1° C., and generallydoes not fluctuate by about, or by more than about, 0.5° C., 0.4° C.,0.3° C., or 0.2° C.), following a specified treatment, such as atreatment designed to reduce fever such as an antipyretic. Anantipyretic may include any agent, e.g., compound, composition, oringredient, that reduces fever, such as one of any number of agentsknown to have antipyretic effects, such as NSAIDs (such as ibuprofen,naproxen, ketoprofen, and nimesulide), salicylates, such as aspirin,choline salicylate, magnesium salicylate, and sodium salicylate,paracetamol, acetaminophen, Metamizole, Nabumetone, Phenaxone,antipyrine, febrifuges. In some embodiments, the antipyretic isacetaminophen. In some embodiments, it is or comprises ibuprophen oraspirin. For example, a subject is considered to have a sustained feverif he or she exhibits or is determined to exhibit a fever of at least ator about 38 or 39 degrees Celsius, which is not reduced by or is notreduced by more than at or about 0.5° C., 0.4° C., 0.3° C., or 0.2° C.,or by at or about 1%, 2%, 3%, 4%, or 5%, over a period of 6 hours, overa period of 8 hours, or over a period of 12 hours, or over a period of24 hours, even following treatment with the antipyretic such as tylenol.In some embodiments, the dosage of the antipyretic is a dosageordinarily effective in such as subject to reduce fever or fever of aparticular type such as fever associated with a bacterial or viralinfection, e.g., a localized or systemic infection.

In some embodiments, severe CRS or grade 3 CRS encompasses an increasein alanine aminotransferase, an increase in aspartate aminotransferase,chills, febrile neutropenia, headache, left ventricular dysfunction,encephalopathy, hydrocephalus, and/or tremor.

In some aspects, the toxic outcome of a therapy, such as a cell therapy,is or is associated with or indicative of neurotoxicity or severeneurotoxicity. In some embodiments, symptoms associated with a clinicalrisk of neurotoxicity include confusion, delirium, expressive aphasia,obtundation, myoclonus, lethargy, altered mental status, convulsions,seizure-like activity, seizures (optionally as confirmed byelectroencephalogram [EEG]), elevated levels of beta amyloid (A),elevated levels of glutamate, and elevated levels of oxygen radicals. Insome embodiments, neurotoxicity is graded based on severity (e.g., usinga Grade 1-5 scale (see, e.g., Guido Cavaletti & Paola Marmiroli NatureReviews Neurology 6, 657-666 (December 2010); National CancerInstitute-Common Toxicity Criteria version 4.03 (NCI-CTCAE v4.03).

In some instances, neurologic symptoms may be the earliest symptoms ofsCRS. In some embodiments, neurologic symptoms are seen to begin 5 to 7days after cell therapy infusion. In some embodiments, duration ofneurologic changes may range from 3 to 19 days. In some cases, recoveryof neurologic changes occurs after other symptoms of sCRS have resolved.In some embodiments, time or degree of resolution of neurologic changesis not hastened by treatment with anti-IL-6 and/or steroid(s).

In some embodiments, a subject is deemed to develop “severeneurotoxicity” in response to, or secondary to, administration of a celltherapy or dose of cells thereof, if, following administration, thesubject displays symptoms that limit self-care (e.g. bathing, dressingand undressing, feeding, using the toilet, taking medications) fromamong: 1) symptoms of peripheral motor neuropathy, includinginflammation or degeneration of the peripheral motor nerves; 2) symptomsof peripheral sensory neuropathy, including inflammation or degenerationof the peripheral sensory nerves, dysesthesia, such as distortion ofsensory perception, resulting in an abnormal and unpleasant sensation,neuralgia, such as intense painful sensation along a nerve or a group ofnerves, and/or paresthesia, such as functional disturbances of sensoryneurons resulting in abnormal cutaneous sensations of tingling,numbness, pressure, cold and warmth in the absence of stimulus. In someembodiments, severe neurotoxicity includes neurotoxicity with a grade of3 or greater, such as set forth in Table 2. In some embodiments, asevere neurotoxicity is deemed to be a prolonged grade 3 if symptoms orgrade 3 neurotoxicity last for 10 days or longer.

TABLE 2 Exemplary Grading Criteria for neurotoxicity Grade Descriptionof Symptoms 1 Mild or asymptomatic symptoms Asymptomatic or Mild 2Presence of symptoms that limit instrumental activities of Moderatedaily living (ADL), such as preparing meals, shopping for groceries orclothes, using the telephone, managing money 3 Presence of symptoms thatlimit self-care ADL, such as Severe bathing, dressing and undressing,feeding self, using the toilet, taking medications 4 Symptoms that arelife-threatening, requiring urgent Life- intervention threatening 5Death Fatal

In some embodiments, the provided embodiments, including methods oftreatment, lead to reduced symptoms associated with neurotoxicityfollowing cell therapy compared to other methods. For example, subjectstreated according to the provided methods may have reduced symptoms ofneurotoxicity, such as limb weakness or numbness, loss of memory,vision, and/or intellect, uncontrollable obsessive and/or compulsivebehaviors, delusions, headache, cognitive and behavioral problemsincluding loss of motor control, cognitive deterioration, and autonomicnervous system dysfunction, and sexual dysfunction, compared to subjectstreated by other methods. In some embodiments, subjects treatedaccording to the provided methods may have reduced symptoms associatedwith peripheral motor neuropathy, peripheral sensory neuropathy,dysesthesia, neuralgia or paresthesia.

In some embodiments, the methods reduce outcomes associated withneurotoxicity including damages to the nervous system and/or brain, suchas the death of neurons. In some aspects, the methods reduce the levelof factors associated with neurotoxicity such as beta amyloid (Aβ),glutamate, and oxygen radicals.

B. Agents for Ameliorating or Treating Toxicity

In some embodiments, the provided methods and articles of manufacturecan be used in connection with, or involve or include, one or moreagents or treatments for treating, preventing, delaying, or attenuatingthe development of a toxicity. In some examples, the agent or othertreatment capable of treating, preventing, delaying, or attenuating thedevelopment of a toxicity is administered prior to and/or concurrentlywith administration of a therapeutic cell composition comprising thegenetically engineered cells.

In some embodiments, the agent, e.g., a toxicity-targeting agent, ortreatment capable of treating, preventing, delaying, or attenuating thedevelopment of a toxicity is a steroid, is an antagonist or inhibitor ofa cytokine receptor, such as IL-6 receptor, CD122 receptor (IL-2Rbetareceptor), or CCR2, or is an inhibitor of a cytokine, such as IL-6,MCP-1, IL-10, IFN-γ, IL-8, or IL-18. In some embodiments, the agent isan agonist of a cytokine receptor and/or cytokine, such as TGF-β. Insome embodiments, the agent, e.g., agonist, antagonist or inhibitor, isan antibody or antigen-binding fragment, a small molecule, a protein orpeptide, or a nucleic acid.

In some embodiments, a fluid bolus can be employed as an intervention,such as to treat hypotension associated with CRS. In some embodiments,the target hematocrit levels are >24%. In some embodiments, theintervention includes the use of absorbent resin technology with bloodor plasma filtration. In some cases, the intervention includes dialysis,plasmapheresis, or similar technologies. In some embodiments,vassopressors or acetaminophen can be employed.

In some embodiments, the agent can be administered sequentially,intermittently, or at the same time as or in the same composition as thetherapy, such as cells for adoptive cell therapy. For example, the agentcan be administered before, during, simultaneously with, or afteradministration of the immunotherapy and/or cell therapy.

In some embodiments, the agent is administered at a time as describedherein and in accord with the provided methods, and/or with the providedarticles of manufacture or compositions. In some embodiments, thetoxicity-targeting agent is administered at a time that is within, suchas less than or no more than, 3, 4, 5, 6, 7, 8, 9 or 10 days afterinitiation of the immunotherapy and/or cell therapy. In someembodiments, the toxicity-targeting agent is administered within orwithin about 1 day, 2 days or 3 days after initiation of administrationof the immunotherapy and/or cell therapy.

In some embodiments, the agent, e.g., toxicity-targeting agent, isadministered to a subject after initiation of administration of theimmunotherapy and/or cell therapy at a time at which the subject doesnot exhibit grade 2 or higher CRS or grade 2 or higher neurotoxicity. Insome aspects, the toxicity-targeting agent is administered afterinitiation of administration of the immunotherapy and/or cell therapy ata time at which the subject does not exhibit severe CRS or severeneurotoxicity. Thus, between initiation of administration of theimmunotherapy and/or cell therapy and the toxicity-targeting agent, thesubject is one that does not exhibit grade 2 or higher CRS, such assevere CRS, and/or does not exhibit grade 2 or higher neurotoxicity,such as severe neurotoxicity.

Non-limiting examples of interventions for treating or ameliorating atoxicity, such as severe CRS (sCRS), are described in Table 3. In someembodiments, the intervention includes tocilizumab or othertoxicity-targeting agent as described, which can be at a time in whichthere is a sustained or persistent fever of greater than or about 38° C.or greater than or greater than about 39° C. in the subject. In someembodiments, the fever is sustained in the subject for more than 10hours, more than 12 hours, more than 16 hours, or more than 24 hoursbefore intervention.

TABLE 3 Symptoms related to CRS Suggested Intervention Fever of ≥38.3°C. Acetaminophen (12.5 mg/kg) PO/IV up to every four hours Persistentfever of ≥39° C. Tocilizumab (8-12 mg/kg) IV for 10 hours that isunresponsive to acetaminophen Persistent fever of ≥39° C. Dexamethasone5-10 mg IV/PO up after tocilizumab to every 6-12 hours with continuedfevers Recurrence of symptoms 48 hours Tocilizumab (8-12 mg/kg) IV afterinitial dose of tocilizumab Hypotension Fluid bolus, targethematocrit >24% Persistent/recurrent hypotension Tocilizumab (8-12mg/kg) IV after initial fluid bolus (within 6 hours) Use of low dosepressors for Dexamethasone 5-10 mg IV/PO up hypotension for longer than12 to every 6 hours with continued hours use of pressors Initiation ofhigher dose Dexamethasone 5-10 mg IV/PO up pressors or addition of a toevery 6 hours with continued second pressor for hypotension use ofpressors Initiation of oxygen Tocilizumab (8-12 mg/kg) IVsupplementation Increasing respiratory support Dexamethasone 5-10 mgIV/PO up with concern for impending to every 6 hours with continuedintubation use of pressors Recurrence/Persistence of Tocilizumab (8-12mg/kg) IV symptoms for which tocilizumab was given ≥48 hours afterinitial dose was administered

In some cases, the agent or therapy or intervention, e.g.,toxicity-targeting agent, is administered alone or is administered aspart of a composition or formulation, such as a pharmaceuticalcomposition or formulation, as described herein. Thus, the agent aloneor as part of a pharmaceutical composition can be administeredintravenously or orally, or by any other acceptable known route ofadministration or as described herein.

In some embodiments, the dosage of agent or the frequency ofadministration of the agent in a dosage regimen is reduced compared tothe dosage of the agent or its frequency in a method in which a subjectis treated with the agent after grade 2 or higher CRS or neurotoxicity,such as after severe, e.g., grade 3 or higher, CRS or after severe,e.g., grade 3 or higher neurotoxicity, has developed or been diagnosed(e.g. after physical signs or symptoms of grade 3 or higher CRS orneurotoxicity has manifested). In some embodiments, the dosage of agentor the frequency of administration of the agent in a dosage regimen isreduced compared to the dosage of the agent or its frequency in a methodin which a subject is treated for CRS or neurotoxicity greater than 3days, 4 days, 5 days, 6 days, 1 week, 2 weeks, three weeks, or moreafter administration of the immunotherapy and/or cell therapy. In someembodiments, the dosage is reduced by greater than or greater than about1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold,8-fold, 9-fold, 10-fold or more. In some embodiments, the dosage isreduced by greater than or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, or more. In some embodiments, the frequency of dosing is reduced,such as the number of daily doses is reduced or the number of days ofdosing is reduced.

1. Steroid

In some embodiments, the agent, e.g., toxicity-targeting agent, thattreats and/or that prevents, delays, or attenuates the development of orrisk for developing a toxicity to an immunotherapy and/or a celltherapy, is a steroid, e.g., corticosteroid. Corticosteroids typicallyinclude glucocorticoids and mineralocorticoids.

Any corticosteroid, e.g., glucocorticoid, can be used in the methodsprovided herein. In some embodiments, glucocorticoids include syntheticand non-synthetic glucocorticoids. Exemplary glucocorticoids include,but are not limited to: alclomethasones, algestones, beclomethasones(e.g. beclomethasone dipropionate), betamethasones (e.g. betamethasone17-valerate, betamethasone sodium acetate, betamethasone sodiumphosphate, betamethasone valerate), budesonides, clobetasols (e.g.clobetasol propionate), clobetasones, clocortolones (e.g. clocortolonepivalate), cloprednols, corticosterones, cortisones and hydrocortisones(e.g. hydrocortisone acetate), cortivazols, deflazacorts, desonides,desoximethasones, dexamethasones (e.g. dexamethasone 21-phosphate,dexamethasone acetate, dexamethasone sodium phosphate), diflorasones(e.g. diflorasone diacetate), diflucortolones, difluprednates,enoxolones, fluazacorts, flucloronides, fludrocortisones (e.g.,fludrocortisone acetate), flumethasones (e.g. flumethasone pivalate),flunisolides, fluocinolones (e.g. fluocinolone acetonide),fluocinonides, fluocortins, fluocortolones, fluorometholones (e.g.fluorometholone acetate), fluperolones (e.g., fluperolone acetate),fluprednidenes, fluprednisolones, flurandrenolides, fluticasones (e.g.fluticasone propionate), formocortals, halcinonides, halobetasols,halometasones, halopredones, hydrocortamates, hydrocortisones (e.g.hydrocortisone 21-butyrate, hydrocortisone aceponate, hydrocortisoneacetate, hydrocortisone buteprate, hydrocortisone butyrate,hydrocortisone cypionate, hydrocortisone hemisuccinate, hydrocortisoneprobutate, hydrocortisone sodium phosphate, hydrocortisone sodiumsuccinate, hydrocortisone valerate), loteprednol etabonate,mazipredones, medrysones, meprednisones, methylprednisolones(methylprednisolone aceponate, methylprednisolone acetate,methylprednisolone hemisuccinate, methylprednisolone sodium succinate),mometasones (e.g., mometasone furoate), paramethasones (e.g.,paramethasone acetate), prednicarbates, prednisolones (e.g. prednisolone25-diethylaminoacetate, prednisolone sodium phosphate, prednisolone21-hemisuccinate, prednisolone acetate; prednisolone farnesylate,prednisolone hemisuccinate, prednisolone-21 (beta-D-glucuronide),prednisolone metasulphobenzoate, prednisolone steaglate, prednisolonetebutate, prednisolone tetrahydrophthalate), prednisones, prednivals,prednylidenes, rimexolones, tixocortols, triamcinolones (e.g.triamcinolone acetonide, triamcinolone benetonide, triamcinolonehexacetonide, triamcinolone acetonide 21-palmitate, triamcinolonediacetate). These glucocorticoids and the salts thereof are discussed indetail, for example, in Remington's Pharmaceutical Sciences, A. Osol,ed., Mack Pub. Co., Easton, Pa. (16th ed. 1980).

In some examples, the glucocorticoid is selected from among cortisones,dexamethasones, hydrocortisones, methylprednisolones, prednisolones andprednisones. In a particular example, the glucocorticoid isdexamethasone.

In some embodiments, the agent is a corticosteroid and is administeredin an amount that is therapeutically effective to treat, ameliorate orreduce one or more symptoms of a toxicity to an immunotherapy and/or acell therapy, such as CRS or neurotoxicity. In some embodiments,indicators of improvement or successful treatment include determinationof the failure to manifest a relevant score on toxicity grading scale(e.g. CRS or neurotoxicity grading scale), such as a score of less than3, or a change in grading or severity on the grading scale as discussedherein, such as a change from a score of 4 to a score of 3, or a changefrom a score of 4 to a score of 2, 1 or 0.

In some aspects, the corticosteroid is provided in a therapeuticallyeffective dose. Therapeutically effective concentration can bedetermined empirically by testing in known in vitro or in vivo (e.g.animal model) systems. For example, the amount of a selectedcorticosteroid to be administered to ameliorate symptoms or adverseeffects of a toxicity to an immunotherapy and/or a cell therapy, such asCRS or neurotoxicity, can be determined by standard clinical techniques.In addition, animal models can be employed to help identify optimaldosage ranges. The precise dosage, which can be determined empirically,can depend on the particular therapeutic preparation, the regime anddosing schedule, the route of administration and the seriousness of thedisease.

The corticosteroid can be administered in any amount that is effectiveto ameliorate one or more symptoms associated with the toxicity, such aswith the CRS or neurotoxicity. The corticosteroid, e.g., glucocorticoid,can be administered, for example, at an amount between at or about 0.1and 100 mg, per dose, 0.1 to 80 mg, 0.1 to 60 mg, 0.1 to 40 mg, 0.1 to30 mg, 0.1 to 20 mg, 0.1 to 15 mg, 0.1 to 10 mg, 0.1 to 5 mg, 0.2 to 40mg, 0.2 to 30 mg, 0.2 to 20 mg, 0.2 to 15 mg, 0.2 to 10 mg, 0.2 to 5 mg,0.4 to 40 mg, 0.4 to 30 mg, 0.4 to 20 mg, 0.4 to 15 mg, 0.4 to 10 mg,0.4 to 5 mg, 0.4 to 4 mg, 1 to 20 mg, 1 to 15 mg or 1 to 10 mg, to a 70kg adult human subject. Typically, the corticosteroid, such as aglucocorticoid is administered at an amount between at or about 0.4 and20 mg, for example, at or about 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.75 mg,0.8 mg, 0.9 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg or 20mg per dose, to an average adult human subject.

In some embodiments, the corticosteroid can be administered, forexample, at a dosage of at or about 0.001 mg/kg (of the subject), 0.002mg/kg, 0.003 mg/kg, 0.004 mg/kg, 0.005 mg/kg, 0.006 mg/kg, 0.007 mg/kg,0.008 mg/kg, 0.009 mg/kg, 0.01 mg/kg, 0.015 mg/kg, 0.02 mg/kg, 0.025mg/kg, 0.03 mg/kg, 0.035 mg/kg, 0.04 mg/kg, 0.045 mg/kg, 0.05 mg/kg,0.055 mg/kg, 0.06 mg/kg, 0.065 mg/kg, 0.07 mg/kg, 0.075 mg/kg, 0.08mg/kg, 0.085 mg/kg, 0.09 mg/kg, 0.095 mg/kg, 0.1 mg/kg, 0.15 mg/kg, 0.2mg/kg, 0.25 mg/kg, 0.30 mg/kg, 0.35 mg/kg, 0.40 mg/kg, 0.45 mg/kg, 0.50mg/kg, 0.55 mg/kg, 0.60 mg/kg, 0.65 mg/kg, 0.70 mg/kg, 0.75 mg/kg, 0.80mg/kg, 0.85 mg/kg, 0.90 mg/kg, 0.95 mg/kg, 1 mg/kg, 1.05 mg/kg, 1.1mg/kg, 1.15 mg/kg, 1.20 mg/kg, 1.25 mg/kg, 1.3 mg/kg, 1.35 mg/kg or 1.4mg/kg, to an average adult human subject, typically weighing about 70 kgto 75 kg.

The corticosteroid, or glucocorticoid, for example dexamethasone, can beadministered orally (tablets, liquid or liquid concentrate), PO,intravenously (IV), intramuscularly or by any other known route or routedescribed herein (e.g., with respect to pharmaceutical formulations). Insome aspects, the corticosteroid is administered as a bolus, and inother aspects it may be administered over a period of time.

In some aspects, the glucocorticoid can be administered over a period ofmore than one day, such as over two days, over 3 days, or over 4 or moredays. In some embodiments, the corticosteroid can be administered oneper day, twice per day, or three times or more per day. For example, thecorticosteroid, e.g., dexamethasone, may in some examples beadministered at 10 mg (or equivalent) IV twice a day for three days.

In some embodiments, the dosage of corticosteroid, e.g., glucocorticoid,is administered in successively lower dosages per treatment. Hence, insome such treatment regimes, the dose of corticosteroid is tapered. Forexample, the corticosteroid may be administered at an initial dose (orequivalent dose, such as with reference to dexamethasone) of 4 mg, andupon each successive administration the dose may be lowered, such thatthe dose is 3 mg for the next administration, 2 mg for the nextadministration, and 1 mg for the next administration

Generally, the dose of corticosteroid administered is dependent upon thespecific corticosteroid, as a difference in potency exists betweendifferent corticosteroids. It is typically understood that drugs vary inpotency, and that doses can therefore vary, in order to obtainequivalent effects. Table 4 shows equivalence in terms of potency forvarious glucocorticoids and routes of administration. Equivalent potencyin clinical dosing is well known. Information relating to equivalentsteroid dosing (in a non-chronotherapeutic manner) may be found in theBritish National Formulary (BNF) 37, March 1999.

TABLE 4 Glucocorticoid administration Glucocorticoid (Route) EquivalencyPotency Hydrocortisone (IV or PO) 20 Prednisone 5 Prednisolone (IV orPO) 5 Methylprednisolone sodium succinate (IV) 4 Dexamethasone (IV orPO) 0.5-0.75

Thus, in some embodiments, the steroid is administered in an equivalentdosage amount of from or from about 1.0 mg to 20 mg dexamethasone perday, such as 1.0 mg to 15 mg dexamethasone per day, 1.0 mg to 10 mgdexamethasone per day, 2.0 mg to 8 mg dexamethasone per day, or 2.0 mgto 6.0 mg dexamethasone per day, each inclusive. In some cases, thesteroid is administered in an equivalent dose of at or about 4 mg or ator about 8 mg dexamethasone per day.

In some embodiments, the steroid is administered if fever persists aftertreatment with tocilizumab. For example, in some embodiments,dexamethasone is administered orally or intravenously at a dosage of5-10 mg up to every 6-12 hours with continued fevers. In someembodiments, tocilizumab is administered concurrently with or subsequentto oxygen supplementation.

2. Microglial Cell Inhibitor

In some embodiments, the inhibitor in the combination therapy is aninhibitor of a microglial cell activity. In some embodiments, theadministration of the inhibitor modulates the activity of microglia. Insome embodiments, the inhibitor is an antagonist that inhibits theactivity of a signaling pathway in microglia. In some embodiments, themicroglia inhibitor affects microglial homeostasis, survival, and/orproliferation. In some embodiments, the inhibitor targets the CSF1Rsignaling pathway. In some embodiments, the inhibitor is an inhibitor ofCSF1R. In some embodiments, the inhibitor is a small molecule. In somecases, the inhibitor is an antibody.

In some aspects, administration of the inhibitor results in one or moreeffects selected from an alteration in microglial homeostasis andviability, a decrease or blockade of microglial cell proliferation, areduction or elimination of microglial cells, a reduction in microglialactivation, a reduction in nitric oxide production from microglia, areduction in nitric oxide synthase activity in microglia, or protectionof motor neurons affected by microglial activation. In some embodiments,the agent alters the level of a serum or blood biomarker of CSF1Rinhibition, or a decrease in the level of urinary collagen type 1cross-linked N-telopeptide (NTX) compared to at a time just prior toinitiation of the administration of the inhibitor. In some embodiments,the administration of the agent transiently inhibits the activity ofmicroglia activity and/or wherein the inhibition of microglia activityis not permanent. In some embodiments, the administration of the agenttransiently inhibits the activity of CSF1R and/or wherein the inhibitionof CSF1R activity is not permanent.

In some embodiments, the agent that reduces microglial cell activity isa small molecule, peptide, protein, antibody or antigen-binding fragmentthereof, an antibody mimetic, an aptamer, or a nucleic acid molecule. Insome embodiments, the method involves administration of an inhibitor ofmicroglia activity. In some embodiments, the agent is an antagonist thatinhibits the activity of a signaling pathway in microglia. In someembodiments, the agent that reduces microglial cell activity affectsmicroglial homeostasis, survival, and/or proliferation.

In some embodiments, the agent that reduces microglial cell activationis selected from an anti-inflammatory agent, an inhibitor of NADPHoxidase (NOX2), a calcium channel blocker, a sodium channel blocker,inhibits GM-CSF, inhibits CSF1R, specifically binds CSF-1, specificallybinds IL-34, inhibits the activation of nuclear factor kappa B (NF-xB),activates a CB₂ receptor and/or is a CB₂ agonist, a phosphodiesteraseinhibitor, inhibits microRNA-155 (miR-155), upregulates microRNA-124(miR-124), inhibits nitric oxide production in microglia, inhibitsnitric oxide synthase, or activates the transcription factor NRF2 (alsocalled nuclear factor (erythroid-derived 2)-like 2, or NFE2L2).

In some embodiments, the agent that reduces microglial cell activitytargets CSF1 (also called macrophage colony-stimulating factor MCSF). Insome embodiments, the agent that reduces microglial cell activityaffects MCSF-stimulated phosphorylation of the M-CSF receptor (Pryer etal. Proc Am Assoc Cancer Res, AACR Abstract nr DDT02-2 (2009)). In somecases, the agent that reduces microglial cell activity is MCS110(international patent application publication number WO2014001802;Clinical Trial Study Record Nos.: A1 NCT00757757; NCT02807844;NCT02435680; NCT01643850).

In some embodiments, the agent that reduces microglial cell activity isa small molecule that targets the CSF1 pathway. In some embodiments, theagent is a small molecule that binds CSF1R. In some embodiments, theagent is a small molecule which inhibits CSF1R kinase activity bycompeting with ATP binding to CSF1R kinase. In some embodiments, theagent is a small molecule which inhibits the activation of the CFS1Rreceptor. In some cases, the binding of the CSF-1 ligand to the CSF1R isinhibited. In some embodiments, the agent that reduces microglial cellactivity is any of the inhibitors described in US Patent ApplicationPublication Number US20160032248.

In some embodiments, the agent is a small molecule inhibitor selectedfrom PLX-3397, PLX7486, JNJ-40346527, JNJ28312141, ARRY-382, PLX73086(AC-708), DCC-3014, AZD6495, GW2580, Ki20227, BLZ945, PLX647, PLX5622.In some embodiments, the agent is any of the inhibitors described inConway et al., Proc Natl Acad Sci USA, 102(44):16078-83 (2005); Dagheret al., Journal of Neuroinflammation, 12:139 (2015); Ohno et al., MolCancer Ther. 5(11):2634-43 (2006); von Tresckow et al., Clin CancerRes., 21(8) (2015); Manthey et al. Mol Cancer Ther. (8(11):3151-61(2009); Pyonteck et al., Nat Med. 19(10): 1264-1272 (2013); Haegel etal., Cancer Res AACR Abstract nr 288 (2015); Smith et al., Cancer ResAACR Abstract nr 4889 (2016); Clinical Trial Study Record Nos.:NCT01525602; NCT02734433; NCT02777710; NCT01804530; NCT01597739;NCT01572519; NCT01054014; NCT01316822; NCT02880371; NCT02673736;international patent application publication numbers WO2008063888A2,WO2006009755A2, US patent application publication numbers US20110044998,US 2014/0065141, and US 2015/0119267.

In some embodiments, the agent that reduces microglial cell activity is4-((2-(((1R,2R)-2-hydroxycyclohexyl)amino)benzo[d]thiazol-6-yl)oxy)-N-methylpicolinamide(BLZ945) or a pharmaceutically acceptable salt thereof or derivativesthereof. In some embodiments, the agent is the following compound:

-   -   wherein R1 is an alkyl pyrazole or an alkyl carboxamide, and R2        is a hydroxycycloalkyl or a pharmaceutically acceptable salt        thereof.

In some embodiments, the agent that reduces microglial cell activity is5-((5-chloro-1H-pyrrolo[2,3-b]pyridin-3-yl)methyl)-N-((6-(trifluoromethyl)pyridin-3-yl)methyl)pyridin-2-amine,N-[5-[(5-Chloro-1H-pyrrolo[2,3-b]pyridin-3-yl)methyl]-2-pyridinyl]-6-(trifluoromethyl)-3-pyridinemethanamine)(PLX 3397) or a pharmaceutically acceptable salt thereof or derivativesthereof. In some embodiments, the agent is5-(1H-Pyrrolo[2,3-b]pyridin-3-ylmethyl)-N-[[4-(trifluoromethyl)phenyl]methyl]-2-pyridinaminedihydrochloride (PLX647) or a pharmaceutically acceptable salt thereofor derivatives thereof. In some embodiments, the agent that reducesmicroglial cell activity is the following compound:

-   -   or a pharmaceutically acceptable salt thereof. In some        embodiments, the agent that reduces microglial cell activity is        the following compound:

or a pharmaceutically acceptable salt thereof. In some embodiments, theagent is any of the inhibitors described in U.S. Pat. No. 7,893,075.

In some embodiments, the agent that reduces microglial cell activity is4-cyano-N-[2-(1-cyclohexen-1-yl)-4-[1-[(dimethylamino)acetyl]-4-piperidinyl]phenyl]-1H-imidazole-2-carboxamidemonohydrochloride (JNJ28312141) or a pharmaceutically acceptable saltthereof or derivatives thereof. In some embodiments, the agent is thefollowing compound:

or a pharmaceutically acceptable salt thereof. In some embodiments, theagent is any of the inhibitors described in U.S. Pat. No. 7,645,755.

In some embodiments, the agent that reduces microglial cell activity is1H-Imidazole-2-carboxamide,5-cyano-N-(2-(4,4-dimethyl-1-cyclohexen-1-yl)-6-(tetrahydro-2,2,6,6-tetramethyl-2H-pyran-4-yl)-3-pyridinyl)-,4-Cyano-1H-imidazole-2-carboxylic acidN-(2-(4,4-dimethylcyclohex-1-enyl)-6-(2,2,6,6-tetramethyltetrahydropyran-4-yl)pyridin-3-yl)amide,4-Cyano-N-(2-(4,4-dimethylcyclohex-1-en-1-yl)-6-(2,2,6,6-tetramethyl-tetrahydro-2H-pyran-4-yl)pyridin-3-yl)-lH-imidazole-2-carboxamide(JNJ-40346527) or a pharmaceutically acceptable salt thereof orderivatives thereof. In some embodiments, the agent is the followingcompound:

or a pharmaceutically acceptable salt thereof.

In another embodiment, the agent that reduces microglial cell activityis 5-(3-Methoxy-4-((4-methoxybenzyl)oxy)benzyl)pyrimidine-2,4-diamine(GW2580) or a pharmaceutically acceptable salt thereof or derivativesthereof. In some embodiments, the agent is the following compound:

or a pharmaceutically acceptable salt thereof (international patentapplication publication number WO2009099553).

In some embodiments, the agent that reduces microglial cell activity is4-(2,4-difluoroanilino)-7-ethoxy-6-(4-methylpiperazin-1-yl)quinoline-3-carboxamide(AZD6495) or a pharmaceutically acceptable salt thereof or derivativesthereof. In some embodiments, the agent is the following compound:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the agent that reduces microglial cell activity isN-{4-[(6,7-dimethoxy-4-quinolyl)oxy]-2-methoxyphenyl}-NO-[1-(1,3-thiazole-2-yl)ethyl]urea(Ki20227) or a pharmaceutically acceptable salt thereof or derivativesthereof. In some embodiments, the agent is the following compound:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the agent that reduces microglial cell activationis an antibody that targets the CSF1 pathway. In some embodiments, theagent is an antibody that binds CSF1R. In some embodiments, theanti-CSF1R antibody blocks CSF1R dimerization. In some embodiments, theanti-CSF1R antibody blocks the CSF1R dimerization interface that isformed by domains D4 and D5 (Ries et al. Cancer Cell 25(6):846-59(2014)). In some cases, the agent is selected from emactuzumab (RG7155;R05509554), Cabiralizumab (FPA-008), LY-3022855 (IMC-CS4), AMG-820,TG-3003, MCS110, H27K15, 12-2D6, 2-4A5 (Rovida and Sbarba, J Clin CellImmunol. 6:6 (2015); Clinical Trial Study Record Nos.: NCT02760797;NCT01494688; NCT02323191; NCT01962337; NCT02471716; NCT02526017;NCT01346358; NCT02265536; NCT01444404; NCT02713529, NCT00757757;NCT02807844; NCT02435680; NCT01643850).

In some embodiments, the agent that reduces microglial cell activationis a tetracycline antibiotic. For example, the agent affects IL-1b,IL-6, TNF-α, or iNOS concentration in microglia cells (Yrjänheikki etal. PNAS 95(26): 15769-15774 (1998); Clinical Trial Study Record No:NCT01120899). In some embodiments, the agent is an opioid antagonist(Younger et al. Pain Med. 10(4):663-672 (2009.) In some embodiments, theagent reduces glutamatergic neurotransmission (U.S. Pat. No. 5,527,814).In some embodiments, the agent modulates NFkB signaling (Valera et al J.Neuroinflammation 12:93 (2015); Clinical Trial Study Record No:NCT00231140). In some embodiments, the agent targets cannabinoidreceptors (Ramirez et al. J. Neurosci 25(8):1904-13(2005)). In someembodiments, the agent is selected from minocycline, naloxone, riluzole,lenalidomide, and a cannabinoid (optionally WIN55 or 212-2).

Nitric oxide production from microglia is believed, in some cases, toresult in or increase neurotoxicity. In some embodiments, the agentmodulates or inhibitis nitric oxide production from microglia. In someembodiments, the agent inhibits nitric oxide synthase (NOS). In someembodiments, the NOS inhibitor is Ronopterin (VAS-203), also known as4-amino-tetrahydrobiopterin (4-ABH4). In some embodiments, the NOSinhibitor is cindunistat, A-84643, ONO-1714, L-NOARG, NCX-456, VAS-2381,GW-273629, NXN-462, CKD-712, KD-7040, or guanidinoethyldisulfide. Insome embodiments, the agent is any of the inhibitors described in Höinget al., Cell Stem Cell. 2012 Nov. 2; 11(5):620-32.

In some embodiments, the agent blocks T cell trafficking, such as to thecentral nervous system. In some embodiments, blocking T cell traffickingcan reduce or prevent immune cells from crossing blood vessel walls intothe central nervous system, including crossing the blood-brain barrier.In some cases, activated antigen-specific T cells produceproinflammatory cytokines, including IFN-γ and TNF, upon reactivation inthe CNS, leading to activation of resident cells such as microglia andastrocytes. See Kivisakk et al., Neurology. 2009 Jun. 2; 72(22):1922-1930. Thus, in some embodiments, sequestering activated T cellsfrom microglial cells, such as by blocking trafficking and/or inhibitingthe ability of such cells to cross the blood-brain barrier, can reduceor eliminate microglial activation. In some embodiments, the agentinhibits adhesion molecules on immune cells, including T cells. In someembodiments, the agent inhibits an integrin. In some embodiments, theintegrin is alpha-4 integrin. In some embodiments, the agent isnatalizumab (Tysabri®). In some embodiments, the agent modulates a cellsurface receptor. In some embodiments, the agent modulates thesphingosine-1-phosphate (SP) receptor, such as S1PR1 or S1PR5. In someembodiments, the agent causes the internalization of a cellularreceptor, such as a sphingosine-1-phosphate (SlP) receptor, such asS1PR1 or S1PR5. In some embodiments, the agent is fingolimod (Gilenya®)or ozanimod (RPC-1063).

The transcription factor NRF2 is believed to regulate the anti-oxidantresponse, for example, by turning on genes that contain a cis-actingelement in their promoter region. An example of such an element includesan antioxidant response element (ARE). In some embodiments, the agentactivates NRF2. In some embodiments, activating NRF2 in microglial cellsreduces the microglial cells' responsiveness to IFN and LPS. In someembodiments, activating NRF2 inhibits, slows, or reduces demyelination,axonal loss, neuronal death, and/or oligodendrocyte death. In someembodiments, the agent upregulates the cellular cytoprotective pathwayregulated by NRF2. In some embodiments, the agent that activates NRF2 isdimethyl fumarate (Tecfidera). In some embodiments, the agent is any ofthe inhibitors described in U.S. Pat. No. 8,399,514. In someembodiments, the agent is any of the inhibitors described in HMing etal., Cell Stem Cell. 2012 Nov. 2; 11(5):620-32.

In some embodiments, the agent that reduces microglial cell activationis(4S,4aS,5aR,12aS)-4,7-bis(dimethylamino)-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide(Minocycline) or a pharmaceutically acceptable sal thereof orderivatives thereof. In some embodiments, the agent is any of thecompounds described in US patent application publication numberUS20100190755. In some embodiments, the agent is the following compound:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the agent that reduces microglial cell activationis 3-(7-amino-3-oxo-1H-isoindol-2-yl)piperidine-2,6-dione (lenalidomide)or a pharmaceutically acceptable salt thereof or derivatives thereof. Insome embodiments, the agent is the following compound:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the agent that reduces microglial cell activationis4R,4aS,7aR,12bS)-4a,9-dihydroxy-3-prop-2-enyl-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-7-one(naloxone) or a phtrmaceutically acceptable salt thereof or derivativesthereof. In some embodiments, the agent is any of the compoundsdescribed in U.S. Pat. No. 8,247,425. In some embodiments, the agent isthe following compound:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the agent that reduces microglial cell activationis 2-amino-6-(trifluoromethoxy)benzothiazole,6-(trifluoromethoxy)benzo[d]thiazol-2-amine, or6-(trifluoromethoxy)-1,3-benzothiazol-2-amine (riluzole) or apharmaceutically acceptable salt thereof or derivatives thereof asdescribed in U.S. Pat. No. 5,527,814. In some embodiments, the agent isthe following compound:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the agent that reduces microglial cell activationis a modulator of a signaling pathway in microglia. In some cases, theagent reduces microglia singling. In some embodiments, the agent is aGM-CSF (CSF2) inhibitor. In other embodiments, the agent that reducesmicroglial cell activation is an ion channel blocker. In some specificembodiments, the agent is a calcium channel blocker. For example, insome specific examples, the agent is a dihydropyridine calcium channelblocker. In some embodiments, the agent is a microRNA inhibitor. Forexample, the agent targets miR-155. In some embodiments, the agent thatreduces microglial cell activation is selected from MOR103, Nimodipine,IVIg, and LNA-anti-miR-155 (Butoxsky et al. Ann Neurol., 77(1):75-99(2015) and Sanz et al., Br J Pharmacol. 167(8): 1702-1711 (2012); Winteret al., Ann Clin and Transl Neurol. 2328-9503 (2016); Clinical TrialStudy Record Nos.: NCT01517282, NCT00750867).

In some embodiments, the agent that reduces microglial cell activationis 3-(2-methoxyethyl) 5-propan-2-yl2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate(nimodipine) or a pharmaceutically acceptable salt thereof orderivatives thereof. In some embodiments, the agent is any of theinhibitors described in U.S. Pat. No. 3,799,934. In some embodiments,the agent is the following compound:

or a pharmaceutically acceptable salt thereof.

In some cases, the agent that reduces microglial cell activation isadministered in a form that only affects to central nervous systemand/or does not affect tumor-associated macrophages. In someembodiments, the agent promotes microglia quiescence but does noteliminate or reduce the number of microglia. In some embodiments, themethod involves inhibiting microglia activity specifically in the brainsuch as described in Ponomarev et al., Nature Medicine, (1):64-70 (2011)

Exemplary agents that reduce microglial cell activation, and exemplarydosing regimens for administering such agents, are set forth in Table 5below.

TABLE 5 Exemplary microglia inhibitors and dosage regimens ExemplaryType of Molecular Inhibitor Molecule Target(s) Exemplary DosingRegimen(s) Pexidartinib small molecule CSF1R; c-Kit; 200 mg tablets,twice daily for 28 days; (PLX3397) FLT3 Administer daily as split doseregimen, five dose-levels possible in dose escalation part: 400 mg 5days on 2 days off (intermittent schedule), 400 mg, 600 mg, 800 mg or1000 mg; 1000 mg/day for 2 weeks then 800 mg/day for 22 weeksEmactuzumab monoclonal CSF1R 100-3000 mg once every 2 weeks (RG1755;antibody RO5509554) Cabiralizumab antibody CSF1R Intravenous infusionover 30 minutes (FPA-008) every 2 weeks LY-3022855 monoclonal CSF1R 1.25mg/kg intravenous delivery every 2 (IMC-CS4) antibody weeks for 6 weeksJNJ-40346527 small molecule CSF1R 100 mg twice daily for 12 weeks; 100-1000 mg capsule daily MCS110 antibody MCSF (CSF1) Up to 4 doses of 10mg/kg MCS110 administered intravenously once every 4 weeks starting atDay 1 MOR103 antibody GM-CSF 6 doses of 0.5-2.0 mg/kg over 70 days IVIgimmunoglobulin Unknown Intravenous infusion of 0.4 g/kg each month for 6months Minocyline small molecule broad spectrum Oral dose of 100 mg ofminocycline antibiotic: IL-1b; twice daily for 24 months IL-6, TNF-a;iNOS Naloxone small molecule Opioid receptors 4.5 mg naltrexonehydrochloride capsules once/day for 8 weeks Lenalidomide/ small moleculeNFkB signaling 100-400 mg daily thalidomide Riluzole small moleculeGlutamate release 50 mg twice daily by microglia Cannabinoids/ smallmolecule cannabinoid Orally 10 mg/kg/day for 6 weeks cannabidiolreceptors (average of 700 mg/day) (e.g. WIN55, 212-2) Dimethyl smallmolecule Nrf2 signaling Starting dose of 120 mg taken orally fumaratetwice/day for 7 days. Dose increased to (Tecfidera ®). 240 mg takenorally twice/day thereafter natalizumab antibody alpha-4 integrin 300 mginfused intravenously over (Tysabri ®) one hour, every four weeksfingolimod small molecule S1P receptors, 0.5 mg orally once-daily(Gilenya ®) including S1PR1 ozanimod small molecule S1PR1 and 0.25 mg,0.5 mg, or 1 mg once daily (RPC-1063) S1PR5

3. Other Agents (e.g. Cytokine Targeting Agents)

In some embodiments, the agent, e.g. toxicity-targeting agent, thattreats or ameliorates symptoms of a toxicity of immunotherapy and/or acell therapy, such as CRS or neurotoxicity, is one that targets acytokine, e.g., is an antagonist or inhibitor of a cytokine, such astransforming growth factor beta (TGF-beta), interleukin 6 (IL-6),interleukin 10 (IL-10), IL-2, MIP1β (CCL4), TNF alpha, IL-1, interferongamma (IFN-gamma), or monocyte chemoattractant protein-1 (MCP-1). Insome embodiments, the agent that treats or ameliorates symptoms of atoxicity of an immunotherapy and/or a cell therapy, such as CRS orneurotoxicity, is one that targets (e.g. inhibits or is an antagonistof) a cytokine receptor, such as IL-6 receptor (IL-6R), IL-2 receptor(IL-2R/CD25), MCP-1 (CCL2) receptor (CCR2 or CCR4), a TGF-beta receptor(TGF-beta I, II, or III), IFN-gamma receptor (IFNGR), MIP10 receptor(e.g., CCR5), TNF alpha receptor (e.g., TNFR1), IL-1 receptor(IL1-Rα/IL-1Rβ), or IL-10 receptor (IL-10R).

The amount of a selected agent that treats or ameliorates symptoms of atoxicity of an immunotherapy and/or a cell therapy, such as CRS orneurotoxicity to be administered to ameliorate symptoms or adverseeffects of a toxicity to an immunotherapy and/or a cell therapy, such asCRS or neurotoxicity, can be determined by standard clinical techniques.Exemplary adverse events include, but are not limited to, an increase inalanine aminotransferase, an increase in aspartate aminotransferase,chills, febrile neutropenia, headache, hypotension, left ventriculardysfunction, encephalopathy, hydrocephalus, seizure, and/or tremor.

In some embodiments, the agent is administered in a dosage amount offrom or from about 30 mg to 5000 mg, such as 50 mg to 1000 mg, 50 mg to500 mg, 50 mg to 200 mg, 50 mg to 100 mg, 100 mg to 1000 mg, 100 mg to500 mg, 100 mg to 200 mg, 200 mg to 1000 mg, 200 mg to 500 mg or 500 mgto 1000 mg.

In some embodiments, the agent is administered from or from about 0.5mg/kg to 100 mg/kg, such as from or from about 1 mg/kg to 50 mg/kg, 1mg/kg to 25 mg/kg, 1 mg/kg to 10 mg/kg, 1 mg/kg to 5 mg/kg, 5 mg/kg to100 mg/kg, 5 mg/kg to 50 mg/kg, 5 mg/kg to 25 mg/kg, 5 mg/kg to 10mg/kg, 10 mg/kg to 100 mg/kg, 10 mg/kg to 50 mg/kg, 10 mg/kg to 25mg/kg, 25 mg/kg to 100 mg/kg, 25 mg/kg to 50 mg/kg to 50 mg/kg to 100mg/kg. In some embodiments, the agent is administered in a dosage amountof from or from about 1 mg/kg to 10 mg/kg, 2 mg/kg to 8 mg/kg, 2 mg/kgto 6 mg/kg, 2 mg/kg to 4 mg/kg or 6 mg/kg to 8 mg/kg, each inclusive. Insome aspects, the agent is administered in a dosage amount of at leastor at least about or about 1 mg/kg, 2 mg/kg, 4 mg/kg, 6 mg/kg, 8 mg/kg,10 mg/kg or more. In some embodiments, the agent is administered at adose of 4 mg/kg or 8 mg/kg.

In some embodiments, the agent is administered by injection, e.g.,intravenous or subcutaneous injections, intraocular injection,periocular injection, subretinal injection, intravitreal injection,trans-septal injection, subscleral injection, intrachoroidal injection,intracameral injection, subconjectval injection, subconjuntivalinjection, sub-Tenon's injection, retrobulbar injection, peribulbarinjection, or posterior juxtascleral delivery. In some embodiments, theyare administered by parenteral, intrapulmonary, and intranasal, and, ifdesired for local treatment, intralesional administration. Parenteralinfusions include intramuscular, intravenous, intraarterial,intraperitoneal, or subcutaneous administration.

In some embodiments, the amount of the agent is administered about orapproximately twice daily, daily, every other day, three times a week,weekly, every other week or once a month.

In some embodiments, the agent is administered as part of a compositionor formulation, such as a pharmaceutical composition or formulation asdescribed below. Thus, in some cases, the composition comprising theagent is administered as described below. In other aspects, the agent isadministered alone and may be administered by any known acceptable routeof administration or by one described herein, such as with respect tocompositions and pharmaceutical formulations.

In some embodiments, the agent that treats or ameliorates symptoms of atoxicity of the immunotherapy and/or cell therapy, such as CRS orneurotoxicity, is an antibody or antigen binding fragment. In someembodiments, the agent is tocilizumab, siltuximab, sarilumab, olokizumab(CDP6038), elsilimomab, ALD518/BMS-945429, sirukumab (CNTO 136),CPSI-2634, ARGX-109, FE301, or FM101.

In some embodiments, the agent is an antagonist or inhibitor of IL-6 orthe IL-6 receptor (IL-6R). In some aspects, the agent is an antibodythat neutralizes IL-6 activity, such as an antibody or antigen-bindingfragment that binds to IL-6 or IL-6R. For example, in some embodiments,the agent is or comprises tocilizumab (atlizumab) or sarilumab,anti-IL-6R antibodies. In some embodiments, the agent is an anti-IL-6Rantibody described in U.S. Pat. No. 8,562,991. In some cases, the agentthat targets IL-6 is an anti-IL-6 antibody, such as siltuximab,elsilimomab, ALD518/BMS-945429, sirukumab (CNTO 136), CPSI-2634,ARGX-109, FE301, FM101, or olokizumab (CDP6038). In some aspects, theagent may neutralize IL-6 activity by inhibiting the ligand-receptorinteractions. The feasibility of this general type of approach has beendemonstrated with a natural occurring receptor antagonist forinterleukin-1. See Harmurn, C. H. et al., Nature (1990) 343:336-340. Insome aspects, the IL-6/IL-6R antagonist or inhibitor is an IL-6 mutein,such as one described in U.S. Pat. No. 5,591,827. In some embodiments,the agent that is an antagonist or inhibitor of IL-6/IL-6R is a smallmolecule, a protein or peptide, or a nucleic acid.

In some embodiments, the agent is tocilizumab. In some embodiments,tocilizumab is administered as an early intervention in accord with theprovided methods, and/or with the provided articles of manufacture orcompositions, at a dosage of from or from about 1 mg/kg to 12 mg/kg,such as at or about 4 mg/kg, 8 mg/kg, or 10 mg/kg. In some embodiments,tocilizumab is administered by intravenous infusion. In someembodiments, tocilizumab is administered for a persistent fever ofgreater than 39° C. lasting 10 hours that is unresponsive toacetaminophen. In some embodiments, a second administration oftocilizumab is provided if symptoms recur after 48 hours of the initialdose.

In some embodiments, the agent is an agonist or stimulator of TGF-β or aTGF-β receptor (e.g., TGF-β receptor I, II, or III). In some aspects,the agent is an antibody that increases TGF-β activity, such as anantibody or antigen-binding fragment that binds to TGF-β or one of itsreceptors. In some embodiments, the agent that is an agonist orstimulator of TGF-β and/or its receptor is a small molecule, a proteinor peptide, or a nucleic acid.

In some embodiments, the agent is an antagonist or inhibitor of MCP-1(CCL2) or a MCP-1 receptor (e.g., MCP-1 receptor CCR2 or CCR4). In someaspects, the agent is an antibody that neutralizes MCP-1 activity, suchas an antibody or antigen-binding fragment that binds to MCP-1 or one ofits receptors (CCR2 or CCR4). In some embodiments, the MCP-1 antagonistor inhibitor is any described in Gong et al. J Exp Med. 1997 Jul. 7;186(1): 131-137 or Shahrara et al. J Immunol 2008; 180:3447-3456. Insome embodiments, the agent that is an antagonist or inhibitor of MCP-1and/or its receptor (CCR2 or CCR4) is a small molecule, a protein orpeptide, or a nucleic acid.

In some embodiments, the agent is an antagonist or inhibitor of IFN-γ oran IFN-γ receptor (IFNGR). In some aspects, the agent is an antibodythat neutralizes IFN-γ activity, such as an antibody or antigen-bindingfragment that binds to IFN-γ or its receptor (IFNGR). In some aspects,the IFN-gamma neutralizing antibody is any described in Dobber et al.Cell Immunol. 1995 February; 160(2):185-92 or Ozmen et al. J Immunol.1993 Apr. 1; 150(7):2698-705. In some embodiments, the agent that is anantagonist or inhibitor of IFN-γ/IFNGR is a small molecule, a protein orpeptide, or a nucleic acid.

In some embodiments, the agent is an antagonist or inhibitor of IL-10 orthe IL-10 receptor (IL-10R). In some aspects, the agent is an antibodythat neutralizes IL-10 activity, such as an antibody or antigen-bindingfragment that binds to IL-10 or IL-10R. In some aspects, the IL-10neutralizing antibody is any described in Dobber et al. Cell Immunol.1995 February; 160(2):185-92 or Hunter et al. J Immunol. 2005 Jun. 1;174(11):7368-75. In some embodiments, the agent that is an antagonist orinhibitor of IL-10/IL-10R is a small molecule, a protein or peptide, ora nucleic acid.

In some embodiments, the agent is an antagonist or inhibitor of IL-1 orthe IL-1 receptor (IL-1R). In some aspects, the agent is an IL-1receptor antagonist, which is a modified form of IL-1R, such as anakinra(see, e.g., Fleischmann et al., (2006) Annals of the rheumatic diseases.65(8):1006-12). In some aspects, the agent is an antibody thatneutralizes IL-1 activity, such as an antibody or antigen-bindingfragment that binds to IL-1 or IL-1R, such as canakinumab (see also EP2277543). In some embodiments, the agent that is an antagonist orinhibitor of IL-1/IL-1R is a small molecule, a protein or peptide, or anucleic acid.

In some embodiments, the agent is an antagonist or inhibitor of a tumornecrosis factor (TNF) or a tumor necrosis factor receptor (TNFR). Insome aspects, the agent is an antibody that blocks TNF activity, such asan antibody or antigen-binding fragment that binds to a TNF, such asTNFα, or its receptor (TNFR, e.g., TNFRp55 or TNFRp75). In some aspects,the agent is selected from among infliximab, adalimumab, certolizumabpegol, golimumab and etanercept. In some embodiments, the agent that isan antagonist or inhibitor of TNF/TNFR is a small molecule, a protein orpeptide, or a nucleic acid. In some embodiments, the agent is a smallmolecule that affects TNF, such as lenalidomide (see, e.g., Muller etal. (1999) Bioorganic & Medicinal Chemistry Letters. 9 (11):1625).

In some embodiments, the agent is an antagonist or inhibitor ofsignaling through the Janus kinase (JAK) and two Signal Transducer andActivator of Transcription (STAT) signaling cascade. JAK/STAT proteinsare common components of cytokine and cytokine receptor signaling. Insome embodiments, the agent that is an antagonist or inhibitor ofJAK/STAT, such as ruxolitinib (see, e.g., Mesa et al. (2012) NatureReviews Drug Discovery. 11(2):103-104), tofacitinib (also known asXeljanz, Jakvinus tasocitinib and CP-690550), Baricitinib (also known asLY-3009104, INCB-28050), Filgotinib (G-146034, GLPG-0634), Gandotinib(LY-2784544), Lestaurtinib (CEP-701), Momelotinib (GS-0387, CYT-387),Pacritinib (SB1518), and Upadacitinib (ABT-494). In some embodiments,the agent is a small molecule, a protein or peptide, or a nucleic acid.

In some embodiments, the agent is a kinase inhibitor. In someembodiments, the agent is an inhibitor of Bruton's tyrosine kinase(BTK). In some embodiments, the inhibitor is or comprises ibrutinib oracalabrutinib (see, e.g., Barrett et al., ASH 58th Annual Meeting SanDiego, Calif. Dec. 3-6, 2016, Abstract 654; Ruella et al., ASH 58thAnnual Meeting San Diego, Calif. Dec. 3-6, 2016, Abstract 2159). In someembodiments, the agent is an inhibitor as described in U.S. Pat. Nos.7,514,444; 8,008,309; 8,476,284; 8,497,277; 8,697,711; 8,703,780;8,735,403; 8,754,090; 8,754,091; 8,957,079; 8,999,999; 9,125,889;9,181,257; or 9,296,753.

In some embodiments, a device, such as absorbent resin technology withblood or plasma filtration, can be used to reduce cytokine levels. Insome embodiments, the device used to reduce cytokine levels is aphysical cytokine absorber, such as an extracorporeal cytokine absorber.In some embodiments, a physical cytokine absorber can be used toeliminate cytokines from the bloodstream in an ex vivo, extracorporealmanner. In some embodiments, the agent is a porous polymer. In someembodiments, the agent is CytoSorb (see, e.g., Basu et al. Indian J CritCare Med. (2014) 18(12): 822-824).

IV. COMPOSITIONS AND FORMULATIONS

In Some Embodiments, the Dose of Cells Comprising Cells Engineered toExpress a recombinant antigen receptor, e.g. CAR or TCR, is provided asa composition or formulation, such as a pharmaceutical composition orformulation. Such compositions can be used in accord with the providedmethods, and/or with the provided articles of manufacture orcompositions, such as in the prevention or treatment of diseases,conditions, and disorders, or in detection, diagnostic, and prognosticmethods.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

In some aspects, the choice of carrier is determined in part by theparticular cell or agent and/or by the method of administration.Accordingly, there are a variety of suitable formulations. For example,the pharmaceutical composition can contain preservatives. Suitablepreservatives may include, for example, methylparaben, propylparaben,sodium benzoate, and benzalkonium chloride. In some aspects, a mixtureof two or more preservatives is used. The preservative or mixturesthereof are typically present in an amount of about 0.0001% to about 2%by weight of the total composition. Carriers are described, e.g., byRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).Pharmaceutically acceptable carriers are generally nontoxic torecipients at the dosages and concentrations employed, and include, butare not limited to: buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride; benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as polyethylene glycol(PEG).

Buffering agents in some aspects are included in the compositions.Suitable buffering agents include, for example, citric acid, sodiumcitrate, phosphoric acid, potassium phosphate, and various other acidsand salts. In some aspects, a mixture of two or more buffering agents isused. The buffering agent or mixtures thereof are typically present inan amount of about 0.001% to about 4% by weight of the totalcomposition. Methods for preparing administrable pharmaceuticalcompositions are known. Exemplary methods are described in more detailin, for example, Remington: The Science and Practice of Pharmacy,Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).

The formulation or composition may also contain more than one activeingredient useful for the particular indication, disease, or conditionbeing prevented or treated with the cells or agents, where therespective activities do not adversely affect one another. Such activeingredients are suitably present in combination in amounts that areeffective for the purpose intended. Thus, in some embodiments, thepharmaceutical composition further includes other pharmaceuticallyactive agents or drugs, such as chemotherapeutic agents, e.g.,asparaginase, busulfan, carboplatin, cisplatin, daunorubicin,doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate,paclitaxel, rituximab, vinblastine, vincristine, etc. In someembodiments, the agents or cells are administered in the form of a salt,e.g., a pharmaceutically acceptable salt. Suitable pharmaceuticallyacceptable acid addition salts include those derived from mineral acids,such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric,and sulphuric acids, and organic acids, such as tartaric, acetic,citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic,and arylsulphonic acids, for example, p-toluenesulphonic acid.

The pharmaceutical composition in some embodiments contains agents orcells in amounts effective to treat or prevent the disease or condition,such as a therapeutically effective or prophylactically effectiveamount. Therapeutic or prophylactic efficacy in some embodiments ismonitored by periodic assessment of treated subjects. For repeatedadministrations over several days or longer, depending on the condition,the treatment is repeated until a desired suppression of diseasesymptoms occurs. However, other dosage regimens may be useful and can bedetermined. The desired dosage can be delivered by a single bolusadministration of the composition, by multiple bolus administrations ofthe composition, or by continuous infusion administration of thecomposition.

The agents or cells can be administered by any suitable means, forexample, by bolus infusion, by injection, e.g., intravenous orsubcutaneous injections, intraocular injection, periocular injection,subretinal injection, intravitreal injection, trans-septal injection,subscleral injection, intrachoroidal injection, intracameral injection,subconjectval injection, subconjuntival injection, sub-Tenon'sinjection, retrobulbar injection, peribulbar injection, or posteriorjuxtascleral delivery. In some embodiments, they are administered byparenteral, intrapulmonary, and intranasal, and, if desired for localtreatment, intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. In some embodiments, a given dose isadministered by a single bolus administration of the cells or agent. Insome embodiments, it is administered by multiple bolus administrationsof the cells or agent, for example, over a period of no more than 3days, or by continuous infusion administration of the cells or agent.

For the prevention or treatment of disease, the appropriate dosage maydepend on the type of disease to be treated, the type of agent oragents, the type of cells or recombinant receptors, the severity andcourse of the disease, whether the agent or cells are administered forpreventive or therapeutic purposes, previous therapy, the subject'sclinical history and response to the agent or the cells, and thediscretion of the attending physician. The compositions are in someembodiments suitably administered to the subject at one time or over aseries of treatments.

The cells or agents may be administered using standard administrationtechniques, formulations, and/or devices. Provided are formulations anddevices, such as syringes and vials, for storage and administration ofthe compositions. With respect to cells, administration can beautologous or heterologous. For example, immunoresponsive cells orprogenitors can be obtained from one subject, and administered to thesame subject or a different, compatible subject. Peripheral bloodderived immunoresponsive cells or their progeny (e.g., in vivo, ex vivoor in vitro derived) can be administered via localized injection,including catheter administration, systemic injection, localizedinjection, intravenous injection, or parenteral administration. Whenadministering a therapeutic composition (e.g., a pharmaceuticalcomposition containing a genetically modified immunoresponsive cell oran agent that treats or ameliorates symptoms of neurotoxicity), it willgenerally be formulated in a unit dosage injectable form (solution,suspension, emulsion).

Formulations include those for oral, intravenous, intraperitoneal,subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal,sublingual, or suppository administration. In some embodiments, theagent or cell populations are administered parenterally. The term“parenteral,” as used herein, includes intravenous, intramuscular,subcutaneous, rectal, vaginal, and intraperitoneal administration. Insome embodiments, the agent or cell populations are administered to asubject using peripheral systemic delivery by intravenous,intraperitoneal, or subcutaneous injection.

Compositions in some embodiments are provided as sterile liquidpreparations, e.g., isotonic aqueous solutions, suspensions, emulsions,dispersions, or viscous compositions, which may in some aspects bebuffered to a selected pH. Liquid preparations are normally easier toprepare than gels, other viscous compositions, and solid compositions.Additionally, liquid compositions are somewhat more convenient toadminister, especially by injection. Viscous compositions, on the otherhand, can be formulated within the appropriate viscosity range toprovide longer contact periods with specific tissues. Liquid or viscouscompositions can comprise carriers, which can be a solvent or dispersingmedium containing, for example, water, saline, phosphate bufferedsaline, polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycol) and suitable mixtures thereof.

Sterile injectable solutions can be prepared by incorporating the agentor cells in a solvent, such as in admixture with a suitable carrier,diluent, or excipient such as sterile water, physiological saline,glucose, dextrose, or the like.

The formulations to be used for in vivo administration are generallysterile. Sterility may be readily accomplished, e.g., by filtrationthrough sterile filtration membranes.

V. KITS AND ARTICLES OF MANUFACTURE

Also provided are articles of manufacture and kits containing engineeredcells expressing a recombinant receptor or compositions thereof, andoptionally instructions for use, for example, instructions foradministering, according to the provided methods. In some embodiments,the instructions include methods for predicting and treating a toxicity.In some aspects, the methods can be used to determine if the subject isat risk or likely at risk for developing a toxicity followingadministration of a cell therapy.

Also provided is a kit containing a cell therapy, said cell therapycomprising cells genetically engineered to express a recombinantreceptor; and instructions for administering the cell therapy to asubject having or suspected of having a tumor, wherein the instructionsspecify: (1) assessing, at two different time points, one or morefactors indicative of disease burden in a subject that is a candidatefor treatment with the cell therapy, wherein, at each of the two timepoints, the subject has not yet been administered the cell therapy andthe factors indicative of disease burden comprises a volumetric measureof the tumor or a level or amount of an inflammatory marker in a samplefrom the subject, wherein the fold change in the factor assessed at thetwo different time points indicates the degree of risk or likely risk ofthe subject developing toxicity following administration of the celltherapy to the subject; and (2) (i) if the fold change is at or below athreshold value, administering the cell therapy to the subject, or (ii)administering the cell therapy to the subject, or (iii) administeringthe cell therapy to the subject at an amount, dose, setting, time orfrequency that is based on the fold change. In some embodiments, the kitfurther comprises instructions for assessing the presence, level oramount of at least one of the inflammatory marker in two or more samplesobtained at two or more time points from a subject that is a candidatefor treatment with the cell therapy and determining a fold-change in thelevel or amount of the marker assessed at two of the two or more timepoints, wherein the fold change indicates the degree of risk or likelyrisk of the subject developing toxicity following administration of thecell therapy to the subject.

In some embodiments, the kit further comprises instructions for (i)administering a therapeutic regimen comprising the cell therapy to thesubject if the fold change is at or below a threshold value, (ii)administering the cell therapy to the subject, or (iii) administeringthe therapeutic regimen to the subject at an amount, dose, setting, timeor frequency based on the fold change of the at least one inflammatorymarker.

In some embodiments, provided are articles of manufacture and/or kitsthat include a composition comprising a therapeutically effective amountof any of the engineered cells described herein, and instructions foradministering, to a subject for treating a disease or condition. In someembodiments, the instructions can specify some or all of the elements ofthe methods provided herein. In some embodiments, the instructionsspecify particular instructions for administration of the cells for celltherapy, e.g., doses, timing, selection and/or identification ofsubjects for administration and conditions for administration. In someembodiments, the articles of manufacture and/or kits further comprise anagent for lymphodepleting therapy, and optionally further includesinstructions for administering the lymphodepleting therapy. In someembodiments, the instructions can be included as a label or packageinsert accompanying the compositions for administration.

In some embodiments, the instructions specify the criteria for selectionor identification of subjects for therapy. In some embodiments, suchcriteria include subjects having NHL or sub-type thereof and/or ahigh-risk NHL. In some embodiments, the instructions specify that thesubjects to be treated include subjects having a disease or conditioncharacterized or determined to be aggressive NHL, diffuse large B celllymphoma (DLBCL), primary mediastinal large B cell lymphoma (PMBCL), Tcell/histocyte-rich large B cell lymphoma (TCHRBCL), Burkitt's lymphoma,mantle cell lymphoma (MCL), and/or follicular lymphoma (FL). Inparticular embodiments, the subject to be treated include subjects withaggressive NHL, in particular, with diffuse large B-cell lymphoma(DLBCL), not otherwise specified (NOS) and in some aspects including denovo and transformed from indolent). In some aspects, the subject orpopulation to be treated may include and/or further include subjectswith primary mediastinal B-cell lymphoma (PMBCL) or follicular lymphomagrade 3B (FL3B). In some embodiments, the subject or population to betreated include those subjects having poor performance status. In someaspects, the population to be treated includes, e.g., subjects having anEastern Cooperative Oncology Group Performance Status (ECOG) that isanywhere from 0-2. In other aspects of any of the embodiments, thesubjects to be treated include ECOG 0-1 or do not include ECOG2subjects. In some embodiments, of any of the embodiments, the subjectsto be treated have failed two or more prior therapies. In someembodiments, the subject does not have DLBCL transformed from marginalzone lymphoma (MZL) and chronic lymphocytic leukemia (CLL; Richter's)and/or has a DLBCL characterized as de novo or transformed from anindolent disease. In some embodiments, the subject has mantle celllymphoma (MCL). In some embodiments, the instructions specify theadministration of the cell therapy is for a subject that is or has beenidentified as having a double/triple hit lymphoma, has been identifiedas having a chemorefractory lymphoma, (e.g., chemorefractory DLBCL)and/or that has not achieved complete remission (CR) in response to aprior therapy.

In some embodiments, the instructions specify the dose of cells to beadministered. For example, in some embodiments, the dose specified inthe instructions include a total recombinant receptor (e.g.,CAR)-expressing cells between about 1×10⁶ and 3×10⁸, e.g., in the rangeof about 1×10⁷ to 2×10⁸ such cells, such as 1×10⁷, 5×10⁷, 1×10⁸ or1.5×10⁸ total such cells, or the range between any two of the foregoingvalues. In some embodiments, the patient is administered multiple doses,and each of the doses or the total dose can be within any of theforegoing values.

In some embodiments, the article of manufacture or kit comprises acontainer, optionally a vial comprising a plurality of CD4+ T cellsexpressing a recombinant receptor, and a container, optionally a vialcomprising a plurality of CD8+ T cells expressing a recombinantreceptor. In some embodiments, the article of manufacture or kitcomprises a container, optionally a vial comprising a plurality of CD4+T cells expressing a recombinant receptor, and further comprises, in thesame container, a plurality of CD8+ T cells expressing a recombinantreceptor. In some embodiments, a cryoprotectant is included with thecells. In some aspects the container is a bag.

In some embodiments, the container such as the vial comprises greaterthan or greater than about 10×10⁶ T cells or recombinantreceptor-expressing T cells, greater than or greater than about 15×10⁶ Tcells or recombinant receptor-expressing T cells, greater than orgreater than about 25×10⁶ T cells or recombinant receptor-expressing Tcell. In some aspects, the vial comprises between about 10 million cellsper ml and about 70 million cells per ml, between about 10 million cellsper ml and about 50 million cells per ml, between about 10 million cellsper ml and about 25 million cells per ml, between about 10 million cellsper ml and about 15 million cells per ml, 15 million cells per ml andabout 70 million cells per ml, between about 15 million cells per ml andabout 50 million cells per ml, between about 15 million cells per ml andabout 25 million cells per ml, between about 25 million cells per ml andabout 70 million cells per ml, between about 25 million cells per ml andabout 50 million cells per ml, and between about 50 million cells per mland about 70 million cells per ml.

In some embodiments, the plurality of vials or plurality of cells orunit dose of cells specified for administration, collectively, comprisesa dose of cells comprising from or from about 1×10⁵ to 5×10⁸ totalrecombinant receptor-expressing T cells or total T cells, 1×10⁵ to 1×10⁸total recombinant receptor-expressing T cells or total T cells, from orfrom about 5×10⁵ to 1×10⁷ total recombinant receptor-expressing T cellsor total T cells, or from or from about 1×10⁶ to 1×10⁷ total recombinantreceptor-expressing T cells or total T cells, each inclusive. In someaspects, the article comprises one or more unit dose of the CD4+ andCD8+ cells or of the CD4+receptor+ cells and CD8+receptor+ cells,wherein the unit dose comprises between at or about 1×10⁷ and at orabout 2×10⁸ recombinant receptor-expressing T cells, between at or about5×10⁷ and at or about 1.5×10⁸ recombinant receptor-expressing T cells,at or about 5×10⁷ recombinant receptor-expressing T cells, at or about1×10⁸ recombinant receptor-expressing T cells, or at or about 1.5×10⁸recombinant receptor-expressing T cells, optionally wherein theinformation in the article specifies administration of one or of aplurality of unit doses and/or a volume corresponding to such one orplurality of unit doses. In some cases, the article comprises one ormore unit doses of the CD8+ cells, wherein the dose comprises between ator about 5×10⁶ and at or about 1×10⁸ recombinant receptor-expressingCD8+ T cells, the dose comprises between at or about 1×10⁷ and at orabout 0.75×10⁸ recombinant receptor-expressing CD8+ T cells, the dosecomprises at or about 2.5×10⁷ recombinant receptor-expressing CD8+ Tcells, or the dose comprises at or about 5×10⁷ recombinantreceptor-expressing CD8+ T cells, or the dose comprises at or about0.75×10⁸ recombinant receptor-expressing CD8+ T cells, optionallywherein the information in the article specifies administration of oneor of a plurality of unit doses and/or a volume corresponding to suchone or plurality of unit doses. In some embodiments, the cells in thearticle, collectively, comprise a dose of cells comprising no more than1×10⁸ total recombinant receptor-expressing T cells or total T cells, nomore than 1×10⁷ total recombinant receptor-expressing T cells or total Tcells, no more than 0.5×10⁷ total recombinant receptor-expressing Tcells or total T cells, no more than 1×10⁶ total recombinantreceptor-expressing T cells or total T cells, no more than 0.5×10⁶ totalrecombinant receptor-expressing T cells or total T cells.

In some embodiments, the instructions can specify dosage regimen andtiming of the administration. For example, in some embodiments, theinstructions can specify administering to the subject multiple doses,e.g., two or more doses, of the cells. In some embodiments, theinstructions specify the timing of the multiple doses, e.g., the seconddose being administered approximately 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20 or 21 days after the first dose; and/or thedosage amount in each dose.

In some embodiments, the article of manufacture or kit comprises aplurality of CD4+ T cells expressing a recombinant receptor, andinstructions for administering, to a subject having a disease orcondition, all or a portion of the plurality of CD4+ T cells and furtheradministering CD8+ T cells expressing a recombinant receptor. In someembodiments, the instructions specify administering the CD4+ T cellsprior to administering the CD8+ cells. In some cases, the instructionsspecify administering the CD8+ T cells prior to administering the CD4+cells. In some embodiments, the article of manufacture or kit comprisesa plurality of CD8+ T cells expressing a recombinant receptor, andinstructions for administering, to a subject having a disease orcondition, all or a portion of the plurality of CD8+ T cells and CD4+ Tcells expressing a recombinant receptor. In some embodiments, theinstructions specify dosage regimen and timing of the administration ofthe cells.

In some aspects, the instructions specify administering all or a portionof the CD4+ T cells and the all or a portion of the CD8+ T cells 0 to 12hours apart, 0 to 6 hours apart or 0 to 2 hours apart. In some cases,the instructions specify administering the CD4+ T cells and the CD8+ Tcells no more than 2 hours, no more than 1 hour, no more than 30minutes, no more than 15 minutes, no more than 10 minutes or no morethan 5 minutes apart.

In some embodiments, the instructions specify the dose or number ofcells or cell type(s) and/or a ratio of cell types, e.g., individualpopulations or sub-types, such as the CD4+ to CD8+ ratio. In someembodiments, the populations or sub-types of cells, such as CD8+ andCD4⁺ T cells. For example, in some embodiments, the instructions specifythat the cells are administered at or within a tolerated range of anoutput ratio of multiple cell populations or sub-types, such as CD4+ andCD8+ cells or sub-types, of between at or about 5:1 and at or about 5:1(or greater than about 1:5 and less than about 5:1), or between at orabout 1:3 and at or about 3:1 (or greater than about 1:3 and less thanabout 3:1), such as between at or about 2:1 and at or about 1:5 (orgreater than about 1:5 and less than about 2:1, such as at or about 5:1,4.5:1, 4:1, 3.5:1, 3:1, 2.5:1, 2:1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1,1.4:1, 1.3:1, 1.2:1, 1.1:1, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5,1:1.6, 1:1.7, 1:1.8, 1:1.9: 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, or 1:5.In some aspects, the tolerated difference is within about 1%, about 2%,about 3%, about 4% about 5%, about 10%, about 15%, about 20%, about 25%,about 30%, about 35%, about 40%, about 45%, about 50% of the desiredratio, including any value in between these ranges.

In some embodiments, the articles of manufacture and/or kits furtherinclude one or more additional agents for therapy, e.g., lymphodepletingtherapy and/or combination therapy, as described herein, and optionallyinstructions for administering the additional agents. In some examples,the articles of manufacture may further contain one or more therapeuticagents. In some embodiments, the therapeutic agent is animmunomodulatory agent, a cytotoxic agent, an anti-cancer agent or aradiotherapeutic.

In some embodiments, the articles of manufacture and/or kits furtherinclude one or more agents or treatments for treating, preventing,delaying, reducing or attenuating the development or risk of developmentof a toxicity and/or instructions for the administration of one or moreagents or treatments for treating, preventing, delaying, reducing orattenuating the development or risk of development of a toxicity in thesubject. In some embodiments, the agent is or comprises an anti-IL-6antibody or anti-IL-6 receptor antibody. For example, in someembodiments, the agent or treatment is or comprises an agent selectedfrom among tocilizumab, siltuximab, clazakizumab, sarilumab, olokizumab(CDP6038), elsilimomab, ALD518/BMS-945429, sirukumab (CNTO 136),CPSI-2634, ARGX-109, FE301 and FM101. For example, in some embodiments,the agent or treatment is or comprises one or more of a steroid; anantagonist or inhibitor of a cytokine receptor or cytokine selected fromamong IL-10, IL-10R, IL-6, IL-6 receptor, IFN7, IFNGR, IL-2, IL-2R/CD25,MCP-1, CCR2, CCR4, MIP10, CCR5, TNFalpha, TNFR1, IL-1, andIL-1Ralpha/IL-1beta; or an agent capable of preventing, blocking orreducing microglial cell activity or function.

In some embodiments, the agent capable of preventing, blocking orreducing microglial cell activity or function is selected from ananti-inflammatory agent, an inhibitor of NADPH oxidase (NOX2), a calciumchannel blocker, a sodium channel blocker, inhibits GM-CSF, inhibitsCSF1R, specifically binds CSF-1, specifically binds IL-34, inhibits theactivation of nuclear factor kappa B (NF-κB), activates a CB₂ receptorand/or is a CB₂ agonist, a phosphodiesterase inhibitor, inhibitsmicroRNA-155 (miR-155) or upregulates microRNA-124 (miR-124). In somecases, the agent is selected from minocycline, naloxone, nimodipine,Riluzole, MOR103, lenalidomide, a cannabinoid (optionally WIN55 or212-2), intravenous immunoglobulin (IVIg), ibudilast, anti-miR-155locked nucleic acid (LNA), MCS110, PLX-3397, PLX647, PLX108-D1, PLX7486,JNJ-40346527, JNJ28312141, ARRY-382, AC-708, DCC-3014,5-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl) pyrimidine-2,4-diamine(GW2580), AZD6495, Ki20227, BLZ945, emactuzumab, IMC-CS4, FPA008,LY-3022855, AMG-820 and TG-3003. In some embodiments, the agent is aninhibitor of colony stimulating factor 1 receptor (CSF1R). For example,the agent PLX-3397, PLX647, PLX108-D1, PLX7486, JNJ-40346527,JNJ28312141, ARRY-382, AC-708, DCC-3014,5-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)pyrimidine-2,4-diamine(GW2580), AZD6495, Ki20227, BLZ945 or a pharmaceutical salt or prodrugthereof; emactuzumab, IMC-CS4, FPA008, LY-3022855, AMG-820 and TG-3003or is an antigen-binding fragment thereof, or a combination of any ofthe foregoing.

In some embodiments, the articles of manufacture and/or kits furtherinclude one or more reagents for assaying biological samples, e.g.,biological samples from subjects who are candidates for administrationor who have been administered the therapy, and optionally instructionsfor use of the reagents or assays. In some embodiments, the biologicalsample is or is obtained from a blood, plasma or serum sample. In someembodiments, the reagents can be used prior to the administration of thecell therapy or after the administration of cell therapy, for diagnosticpurposes, to identify subjects and/or to assess treatment outcomesand/or toxicities. For example, in some embodiments, the article ofmanufacture and/or kits further contain reagents for measuring the levelof particular biomarkers, e.g., inflammatory markers, that areassociated with toxicity, and instructions for measuring. In someembodiments, the reagents include components for performing an in vitroassay to measure the inflammatory markers, such as an immunoassay, anaptamer-based assay, a histological or cytological assay, or an mRNAexpression level assay. In some embodiments, the in vitro assay isselected from among an enzyme linked immunosorbent assay (ELISA),immunoblotting, immunoprecipitation, radioimmunoassay (RIA),immunostaining, flow cytometry assay, surface plasmon resonance (SPR),chemiluminescence assay, lateral flow immunoassay, inhibition assay andavidity assay. In some aspects, the reagent is a binding reagent thatspecifically binds the inflammatory markers. In some cases, the bindingreagent is an antibody or antigen-binding fragment thereof, an aptameror a nucleic acid probe.

In some embodiments, the articles of manufacture and/or kits compriseone or more reagent capable of detecting one or more inflammatorymarkers and instructions for using the reagent to assay a biologicalsample from a subject that is a candidate for treatment, wherein the oneor more inflammatory marker is selected from C-reactive protein (CRP),erythrocyte sedimentation rate (ESR), albumin, ferritin, β2microglobulin (β2-M), or lactate dehydrogenase (LDH). In someembodiments, instructions for assaying presence or absence, level,amount, or concentration of an inflammatory marker in the subject at twotime points and determining a fold change in the inflammatory markerbetween the two time points. In some cases, the instructions specifythat the fold change in inflammatory marker is compared to a thresholdfold change of the inflammatory marker.

In some embodiments, the articles of manufacture and/or kits compriseinstructions for measuring a volumetric measure of tumor burden. In somecases, the instructions specify measuring a volumetric measure oftumor(s) such as a sum of the products of diameters (SPD), longest tumordiameters (LD), sum of longest tumor diameters (SLD), tumor volume,necrosis volume, necrosis-tumor ratio (NTR), peritumoral edema (PTE),and edema-tumor ratio (ETR) at two time points prior to administrationof the cell therapy. In some embodiments, instructions are provided forusing imaging techniques to assess tumor burden in the subject at twotime points and determining a fold change in the volumetric measurementof tumor the two time points. In some cases, the instructions specifythat the fold change in volumetric measurement of tumor is compared to athreshold fold change of the volumetric measurement.

In some embodiments, the instructions are included which specify, if thefold change in the level, amount or concentration of the inflammatorymarker in the sample is at or above a threshold fold change level forthe inflammatory marker, administering to the subject an agent or othertreatment capable of treating, preventing, delaying, reducing orattenuating the development or risk of development of a toxicity (i)prior to, (ii) within one, two, or three days of, (iii) concurrentlywith and/or (iv) at first fever following, the initiation ofadministration of the cell therapy to the subject. In some cases, theinstructions specify that if the level, amount or concentration of theinflammatory marker in the sample is at or above a threshold level forthe inflammatory marker, the cell therapy is administered to the subjectat a reduced dose or at a dose that is not associated with risk ofdeveloping toxicity or severe toxicity, or is not associated with a riskof developing a toxicity or severe toxicity in a majority of subjects,and/or a majority of subjects having a disease or condition that thesubject has or is suspected of having, following administration of thecell therapy. In some cases, the instructions specify that if the level,amount or concentration of the inflammatory marker in the sample is ator above a threshold level for the inflammatory marker, the cell therapyis administered in an in-patient setting and/or with admission to thehospital for one or more days, optionally wherein the cell therapy isotherwise to be administered to subjects on an outpatient basis orwithout admission to the hospital for one or more days.

In some embodiments, the instructions for administering the cell therapyspecify, if the level, amount or concentration of the inflammatorymarker in the sample, is below a threshold level, administering to thesubject the cell therapy, optionally at a non-reduced dose, optionallyon an outpatient basis or without admission to the hospital for one ormore days. In some embodiments, the instructions for administering thecell therapy specify, if the level, amount or concentration of theinflammatory marker in the sample, is below a threshold level, prior toor concurrently with administering the cell therapy and/or prior to thedevelopment of a sign or symptom of a toxicity other than fever, anagent or treatment capable of treating, preventing, delaying, orattenuating the development of the toxicity is not administered to thesubject. In some aspects, the instructions for administering the celltherapy specify that if the level, amount or concentration of theinflammatory marker in the sample, is below a threshold level, theadministration of the cell therapy is to be or may be administered tothe subject on an outpatient setting and/or without admission of thesubject to the hospital overnight or for one or more consecutive daysand/or is without admission of the subject to the hospital for one ormore days.

The articles of manufacture and/or kits may further include a celltherapy and/or further include instructions for use with, prior toand/or in connection with treatment with the cell therapy. In someembodiments, the instructions are included for administering the agentand the instructions specify if the fold change in level, amount orconcentration of the inflammatory marker in the sample, is at or above athreshold level administering to the subject the agent. In some aspects,the instructions further specify administering a cell therapy to thesubject, wherein administration of the agent is to be carried out (i)prior to, (ii) within one, two, or three days of, (iii) concurrentlywith and/or (iv) at first fever following, the initiation ofadministration of the cell therapy to the subject.

The articles of manufacture or kits include one or more containers,typically a plurality of containers, packaging material, and a label orpackage insert on or associated with the container or containers and/orpackaging, generally including instructions for administration of thecells to a subject. The articles of manufacture and/or kits may includea container and a label or package insert on or associated with thecontainer.

In some embodiments, the containers contain the cells to beadministered, e.g., one or more unit doses thereof. The article ofmanufacture typically includes a plurality of containers, eachcontaining a single unit dose of the cells. The unit dose may be anamount or number of the cells to be administered to the subject in thefirst dose or twice the number (or more) the cells to be administered inthe first or any one or more consecutive dose(s). It may be the lowestdose or lowest possible dose of the cells that would be administered tothe subject in connection with the administration method. In someembodiments, the unit dose is the minimum number of cells or number ofcells or the minimum number of reference units or the target referenceunits or reference units within a target range that would beadministered in a single dose to any subject having a particular diseaseor condition or any subject, according to the methods herein.

Suitable containers include, for example, bottles, vials, syringes, IVsolution bags, etc. The containers may be formed from a variety ofmaterials such as glass or plastic. The container in some embodimentsholds a composition which is by itself or combined with anothercomposition effective for treating, preventing and/or diagnosing thecondition. In some embodiments, the container has a sterile access port.Exemplary containers include an intravenous solution bags, vials,including those with stoppers pierceable by a needle for injection, orbottles or vials for orally administered agents. The label or packageinsert may indicate that the composition is used for treating a diseaseor condition. The article of manufacture may include (a) a firstcontainer with a composition contained therein, wherein the compositionincludes engineered cells expressing a recombinant receptor; and (b) asecond container with a composition contained therein, wherein thecomposition includes the second agent. In some embodiments, the articleof manufacture may include (a) a first container with a firstcomposition contained therein, wherein the composition includes asubtype of engineered cells expressing a recombinant receptor; and (b) asecond container with a composition contained therein, wherein thecomposition includes a different subtype of engineered cells expressinga recombinant receptor. The article of manufacture may further include apackage insert indicating that the compositions can be used to treat aparticular condition. Alternatively, or additionally, the article ofmanufacture may further include another or the same container comprisinga pharmaceutically-acceptable buffer. It may further include othermaterials such as other buffers, diluents, filters, needles, and/orsyringes.

In particular embodiments, the containers are bags, e.g., flexible bags,such as those suitable for infusion of cells to subjects, e.g., flexibleplastic or PVC bags, and/or IV solution bags. The bags in someembodiments are sealable and/or able to be sterilized, so as to providesterile solution and delivery of the cells and compositions. In someembodiments, the containers, e.g., bags, have a capacity of at or aboutor at least at or about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200,300, 400, 500, or 1000 mL capacity, such as between at or about 10 andat or about 100 or between at or about 10 and at or about 500 mLcapacity, each inclusive. In some embodiments, the containers, e.g.,bags, are and/or are made from material which is stable and/or providestable storage and/or maintenance of cells at one or more of varioustemperatures, such as in cold temperatures, e.g. below at or about or ator about −20° C., −80° C., −120° C., 135° C. and/or temperaturessuitable for cryopreservation, and/or other temperatures, such astemperatures suitable for thawing the cells and body temperature such asat or about 37° C., for example, to permit thawing, e.g., at thesubject's location or location of treatment, e.g., at bedside,immediately prior to treatment.

The containers may be formed from a variety of materials such as glassor plastic. In some embodiments, the container has one or more port,e.g., sterile access ports, for example, for connection of tubing orcannulation to one or more tubes, e.g., for intravenous or otherinfusion and/or for connection for purposes of transfer to and fromother containers, such as cell culture and/or storage bags or othercontainers. Exemplary containers include infusion bags, intravenoussolution bags, vials, including those with stoppers pierceable by aneedle for injection.

The article of manufacture may further include a package insert or labelwith one or more pieces of identifying information and/or instructionsfor use. In some embodiments, the information or instructions indicatesthat the contents can or should be used to treat a particular conditionor disease, and/or providing instructions therefor. The label or packageinsert may indicate that the contents of the article of manufacture areto be used for treating the disease or condition. In some embodiments,the label or package insert provides instructions to treat a subject,e.g., the subject from which the cells have been derived, via a methodinvolving the administration of a first and one or more consecutivedoses of the cells, e.g., according to any of the embodiments of theprovided methods. In some embodiments, the instructions specifyadministration, in a first dose, of one unit dose, e.g., the contents ofa single individual container in the article of manufacture, followed byone or more consecutive doses at a specified time point or within aspecified time window and/or after the detection of the presence orabsence or amount or degree of one or more factors or outcomes in thesubject.

In some embodiments, the instructions specify administering one or moreof the unit doses to the subject.

In some embodiments, the label or package insert or packaging comprisesan identifier to indicate the specific identity of the subject fromwhich the cells are derived and/or are to be administered. In the caseof autologous transfer, the identity of the subject from which the cellsare derived is the same as the identity of the subject to which thecells are to be administered. Thus, the identifying information mayspecify that the cells are to be administered to a particular patient,such as the one from which the cells were originally derived. Suchinformation may be present in the packaging material and/or label in theform of a bar code or other coded identifier, or may indication the nameand/or other identifying characteristics of the subject.

The article of manufacture in some embodiments includes one or more,typically a plurality, of containers containing compositions comprisingthe cells, e.g., individual unit dose forms thereof, and further includeone or more additional containers with a composition contained thereinwhich includes a further agent, such as a cytotoxic or otherwisetherapeutic agent, for example, which is to be administered incombination, e.g., simultaneously or sequentially in any order, with thecells. Alternatively, or additionally, the article of manufacture mayfurther include another or the same container comprising apharmaceutically-acceptable buffer. It may further include othermaterials such as other buffers, diluents, filters, tubing, needles,and/or syringes.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

VI. DEFINITIONS

The terms “polypeptide” and “protein” are used interchangeably to referto a polymer of amino acid residues, and are not limited to a minimumlength. Polypeptides, including the provided receptors and otherpolypeptides, e.g., linkers or peptides, may include amino acid residuesincluding natural and/or non-natural amino acid residues. The terms alsoinclude post-expression modifications of the polypeptide, for example,glycosylation, sialylation, acetylation, and phosphorylation. In someaspects, the polypeptides may contain modifications with respect to anative or natural sequence, as long as the protein maintains the desiredactivity. These modifications may be deliberate, as throughsite-directed mutagenesis, or may be accidental, such as throughmutations of hosts which produce the proteins or errors due to PCRamplification.

As used herein, a “subject” is a mammal, such as a human or otheranimal, and typically is human. In some embodiments, the subject, e.g.,patient, to whom the agent or agents, cells, cell populations, orcompositions are administered, is a mammal, typically a primate, such asa human. In some embodiments, the primate is a monkey or an ape. Thesubject can be male or female and can be any suitable age, includinginfant, juvenile, adolescent, adult, and geriatric subjects. In someembodiments, the subject is a non-primate mammal, such as a rodent.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to complete or partial amelioration orreduction of a disease or condition or disorder, or a symptom, adverseeffect or outcome, or phenotype associated therewith. Desirable effectsof treatment include, but are not limited to, preventing occurrence orrecurrence of disease, alleviation of symptoms, diminishment of anydirect or indirect pathological consequences of the disease, preventingmetastasis, decreasing the rate of disease progression, amelioration orpalliation of the disease state, and remission or improved prognosis.The terms do not imply complete curing of a disease or completeelimination of any symptom or effect(s) on all symptoms or outcomes.

As used herein, “delaying development of a disease” means to defer,hinder, slow, retard, stabilize, suppress and/or postpone development ofthe disease (such as cancer). This delay can be of varying lengths oftime, depending on the history of the disease and/or individual beingtreated. In some embodiments, sufficient or significant delay can, ineffect, encompass prevention, in that the individual does not developthe disease. For example, a late stage cancer, such as development ofmetastasis, may be delayed.

“Preventing,” as used herein, includes providing prophylaxis withrespect to the occurrence or recurrence of a disease in a subject thatmay be predisposed to the disease but has not yet been diagnosed withthe disease. In some embodiments, the provided cells and compositionsare used to delay development of a disease or to slow the progression ofa disease.

As used herein, to “suppress” a function or activity is to reduce thefunction or activity when compared to otherwise same conditions exceptfor a condition or parameter of interest, or alternatively, as comparedto another condition. For example, cells that suppress tumor growthreduce the rate of growth of the tumor compared to the rate of growth ofthe tumor in the absence of the cells.

An “effective amount” of an agent, e.g., a pharmaceutical formulation,cells, or composition, in the context of administration, refers to anamount effective, at dosages/amounts and for periods of time necessary,to achieve a desired result, such as a therapeutic or prophylacticresult.

A “therapeutically effective amount” of an agent, e.g., a pharmaceuticalformulation or cells, refers to an amount effective, at dosages and forperiods of time necessary, to achieve a desired therapeutic result, suchas for treatment of a disease, condition, or disorder, and/orpharmacokinetic or pharmacodynamic effect of the treatment. Thetherapeutically effective amount may vary according to factors such asthe disease state, age, sex, and weight of the subject, and thepopulations of cells administered. In some embodiments, the providedmethods involve administering the cells and/or compositions at effectiveamounts, e.g., therapeutically effective amounts.

A “prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically but not necessarily, since a prophylacticdose is used in subjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount. In the context of lower tumor burden, theprophylactically effective amount in some aspects will be higher thanthe therapeutically effective amount.

The term “about” as used herein refers to the usual error range for therespective value readily known to the skilled person in this technicalfield. Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse.

As used herein, the singular forms “a,” “an,” and “the” include pluralreferents unless the context clearly dictates otherwise. For example,“a” or “an” means “at least one” or “one or more.”

Throughout this disclosure, various aspects of the claimed subjectmatter are presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theclaimed subject matter. Accordingly, the description of a range shouldbe considered to have specifically disclosed all the possible sub-rangesas well as individual numerical values within that range. For example,where a range of values is provided, it is understood that eachintervening value, between the upper and lower limit of that range andany other stated or intervening value in that stated range isencompassed within the claimed subject matter. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the claimed subjectmatter, subject to any specifically excluded limit in the stated range.Where the stated range includes one or both of the limits, rangesexcluding either or both of those included limits are also included inthe claimed subject matter. This applies regardless of the breadth ofthe range.

As used herein, a composition refers to any mixture of two or moreproducts, substances, or compounds, including cells. It may be asolution, a suspension, liquid, powder, a paste, aqueous, non-aqueous orany combination thereof.

As used herein, “enriching” when referring to one or more particularcell type or cell population, refers to increasing the number orpercentage of the cell type or population, e.g., compared to the totalnumber of cells in or volume of the composition, or relative to othercell types, such as by positive selection based on markers expressed bythe population or cell, or by negative selection based on a marker notpresent on the cell population or cell to be depleted. The term does notrequire complete removal of other cells, cell type, or populations fromthe composition and does not require that the cells so enriched bepresent at or even near 100% in the enriched composition.

As used herein, a statement that a cell or population of cells is“positive” for a particular marker refers to the detectable presence onor in the cell of a particular marker, typically a surface marker. Whenreferring to a surface marker, the term refers to the presence ofsurface expression as detected by flow cytometry, for example, bystaining with an antibody that specifically binds to the marker anddetecting said antibody, wherein the staining is detectable by flowcytometry at a level substantially above the staining detected carryingout the same procedure with an isotype-matched control or fluorescenceminus one (FMO) gating control under otherwise identical conditionsand/or at a level substantially similar to that for cell known to bepositive for the marker, and/or at a level substantially higher thanthat for a cell known to be negative for the marker.

As used herein, a statement that a cell or population of cells is“negative” for a particular marker refers to the absence of substantialdetectable presence on or in the cell of a particular marker, typicallya surface marker. When referring to a surface marker, the term refers tothe absence of surface expression as detected by flow cytometry, forexample, by staining with an antibody that specifically binds to themarker and detecting said antibody, wherein the staining is not detectedby flow cytometry at a level substantially above the staining detectedcarrying out the same procedure with an isotype-matched control orfluorescence minus one (FMO) gating control under otherwise identicalconditions, and/or at a level substantially lower than that for cellknown to be positive for the marker, and/or at a level substantiallysimilar as compared to that for a cell known to be negative for themarker.

The term “vector,” as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors.”

VII. EXEMPLARY EMBODIMENTS

Among the provided embodiments are:

1. A method of assessing a risk of a toxicity or a toxicity-relatedoutcome, following administration of a cell therapy, the methodcomprising:

(1) assessing a factor indicative of disease burden at two time pointsprior to receiving a cell therapy from a subject that is a candidate forreceiving a cell therapy for treatment of a disease or condition; and

(2) determining a fold change in the factor indicative of disease burdenbetween the two time points, wherein the fold change indicates the riskor likely risk of the subject developing a toxicity followingadministration of the therapy to the subject.

2. The method of embodiment 1, wherein the factor indicative of diseaseburden is a volumetric measure of a tumor or is an inflammatory markerin a sample from a subject.

3. The method of embodiment 2, wherein the factor indicative of diseaseburden is a volumetric measure and the volumetric measure is a sum ofthe products of diameters (SPD), longest tumor diameters (LD), sum oflongest tumor diameters (SLD), tumor volume, necrosis volume,necrosis-tumor ratio (NTR), peritumoral edema (PTE), and edema-tumorratio (ETR).

4. The method of embodiment 2 or embodiment 3, wherein the volumetricmeasure is measured using computed tomography (CT), positron emissiontomography (PET), and/or magnetic resonance imaging (MRI) of thesubject.

5. The method of embodiment 2, wherein the factor indicative of diseaseburden is an inflammatory marker and the inflammatory marker isC-reactive protein (CRP), erythrocyte sedimentation rate (ESR), albumin,ferritin, β2 microglobulin (β2-M), or lactate dehydrogenase (LDH).

6. The method of embodiment 2 or embodiment 5, wherein the sample is orcomprises a blood sample, plasma sample, or serum sample.

7. The method of any of embodiments 2, 5 or 6, wherein the inflammatorymarker is assessed using a colorimetric assay or an immunoassay.

8. The method of embodiment 7, wherein the inflammatory marker isassessed using an immunoassay and the immunoassay is selected fromenzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA),radioimmunoassay (RIA), surface plasmon resonance (SPR), Western Blot,Lateral flow assay, immunohistochemistry, protein array or immuno-PCR(iPCR).

9. The method of any of embodiments 1-8, wherein the subject is a human.

10. The method of any of embodiments 1-9, wherein the two time pointscomprises a first time point and a second time point, and wherein thefold change is a ratio of the factor indicative of disease burden at thefirst time point and the second time point.

11. The method of any of embodiments 1-10, wherein the two time pointsare both no more than one month or two months prior to receiving thecell therapy.

12 The method of any of embodiments 1-11, wherein the two time pointsare not less than one week, two weeks, three weeks, four weeks, or fiveweeks apart.

13. The method of any of embodiments 1-12, wherein the two time pointsare not less than three weeks apart.

14. The method of any of embodiments 1-13, wherein the two time pointsare not more than four weeks apart, five weeks, or six weeks apart.

15. The method of any of embodiments 1-14, wherein the second time pointis more than 1, 2, 3, 4, 5, 6, or 7 days before administration of thecell therapy.

16. The method of any of embodiments 1-13, wherein the cell therapycomprises cells engineered to express a recombinant receptor.

17. The method of any of embodiments 1-16, wherein the toxicity isneurotoxicity and/or cytokine release syndrome (CRS).

18. The method of any of embodiments 1-17, wherein the toxicity is earlytoxicity that develops within 7 days of administration of the celltherapy.

19. The method of any of embodiments 1-18, wherein the toxicity developswithin 3, 4, 5, 6, or 7 days of administration of the cell therapy.

20. The method of any of embodiments 1-19, wherein the toxicity is afirst sign of a fever or is a sustained fever following administrationof the cell therapy.

21. The method of any of embodiments 1-20, wherein:

the subject is or is likely at risk of developing toxicity if the foldchange is at or above a threshold value; or

the subject is not or is likely not at risk of developing toxicity ifthe fold change is below a threshold value.

22. The method of embodiment 21, wherein the threshold value is a valuethat:

i) is within 25%, within 20%, within 15%, within 10%, or within 5% abovethe average fold change of the factor indicative of disease burdenand/or is within a standard deviation above the average fold change ofthe factor indicative of disease burden in a plurality of controlsubjects;

ii) is above the highest fold change of the factor indicative of diseaseburden, optionally within 50%, within 25%, within 20%, within 15%,within 10%, or within 5% above such highest fold change, measured in atleast one subject from among a plurality of control subjects; and/or

iii) is above the highest fold change as measured among more than 75%,80%, 85%, 90%, or 95%, or 98% of subjects from a plurality of controlsubjects.

23. The method of embodiment 22, wherein the plurality of controlsubjects are a group of subjects prior to receiving a cell therapy fortreating a disease or condition, said cell therapy containing cellsgenetically engineered with a recombinant receptor, wherein each of thesubjects of the group went on to develop toxicity, optionally earlytoxicity, optionally a fever or a sustained fever, within 7 days afterreceiving the cell therapy for treating the same disease or condition.

24. The method of any of embodiment 1-21, wherein if the subject isindicated as likely to develop toxicity, selecting the subject foradministration of a therapeutic regimen, the therapeutic regimencomprising administering to the subject:

i. an agent or other treatment capable of treating, preventing,delaying, reducing or attenuating the development or risk of developmentof a toxicity and the cell therapy, wherein administration of the agentis to be administered (i) prior to, (ii) within one, two, or three daysof, (iii) concurrently with and/or (iv) at first fever following, theinitiation of administration of the cell therapy to the subject;

ii. the cell therapy at a reduced dose or at a dose that is notassociated with risk of developing toxicity or severe toxicity, or isnot associated with a risk of developing a toxicity or severe toxicityin a majority of subjects, and/or a majority of subjects having adisease or condition that the subject has or is suspected of having,following administration of the cell therapy; and/or

iii. the cell therapy in an in-patient setting and/or with admission tothe hospital for one or more days, optionally wherein the cell therapyis otherwise to be administered to subjects on an outpatient basis orwithout admission to the hospital for one or more days; or

iv. an alternative therapeutic treatment other than the cell therapy.

25. The method of any of embodiments 1-21, wherein if subject isindicated as likely not at risk of developing toxicity, selecting thesubject for administration of a therapeutic regimen, the therapeuticregimen comprising administering to the subject:

i. the cell therapy, optionally at a non-reduced dose, optionally on anoutpatient basis or without admission to the hospital for one or moredays;

ii. the cell therapy, wherein administration of the cell therapy doesnot comprise administering, prior to or concurrently with administeringthe cell therapy and/or prior to the development of a sign or symptom oftoxicity other than fever, an agent or treatment capable of treating,preventing, delaying, or attenuating the development of the toxicity; or

iii. the cell therapy in an outpatient setting and/or without admissionof the subject to the hospital overnight or for one or more consecutivedays and/or is without admission of the subject to the hospital for oneor more days.

26. The method of any of embodiments 1-21, wherein if the subject isindicated as likely to develop toxicity following administration of thecell therapy, the cell therapy is not administered to the subject.

27. The method of embodiment 24 or embodiment 25, further comprisingadministering the therapeutic regimen to the selected subject.

28. A method of treatment, the method comprising administering atherapeutic regimen to a subject that is a candidate for receiving acell therapy for treatment of a disease or condition, wherein theadministration is carried out following or based on the results ofassessing the subject for a fold change in a factor indicative ofdisease burden between two time points prior to receiving a celltherapy.

29. The method of embodiment 28, wherein the factor indicative ofdisease burden is a volumetric measure of a tumor or is an inflammatorymarker in a sample from a subject.

30. The method of embodiment 29, wherein the factor indicative ofdisease burden is a volumetric measure and the volumetric measure is asum of the products of diameters (SPD), longest tumor diameters (LD),sum of longest tumor diameters (SLD), necrosis, tumor volume, necrosisvolume, necrosis-tumor ratio (NTR), peritumoral edema (PTE), andedema-tumor ratio (ETR).

31. The method of embodiment 29 or embodiment 30, wherein the volumetricmeasure is measured using computed tomography (CT), positron emissiontomography (PET), and/or magnetic resonance imaging (MRI) of thesubject.

32. The method of embodiment 29, wherein the factor indicative ofdisease burden is an inflammatory marker and the inflammatory marker isC-reactive protein (CRP), erythrocyte sedimentation rate (ESR), albumin,ferritin, β2 microglobulin (β2-M), or lactate dehydrogenase (LDH).

33. The method of embodiment 29 or embodiment 32, wherein the sample isor comprises a blood sample, plasma sample, or serum sample.

34. The method of any of embodiments 29, 32 or 33, wherein theinflammatory marker is assessed using a colorimetric assay or animmunoassay.

35. The method of embodiment 34, wherein the inflammatory marker isassessed using an immunoassay and the immunoassay is selected fromenzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA),radioimmunoassay (RIA), surface plasmon resonance (SPR), Western Blot,Lateral flow assay, immunohistochemistry, protein array or immuno-PCR(iPCR).

36. The method of any of embodiments 28-34, wherein the subject is ahuman.

37. The method of any of embodiments 28-36, wherein the two time pointscomprises a first time point and a second time point, and wherein thefold change is a ratio of the factor indicative of disease burden at thefirst time point and the second time point.

38. The method of any of embodiments 28-37, wherein the two time pointsare both no more than one month or two months prior to receiving thecell therapy.

39. The method of any of embodiments 28-38, wherein the two time pointsare not less than one week, two weeks, three weeks, four weeks, or fiveweeks apart.

40. The method of any of embodiments 28-39, wherein the two time pointsare not less than three weeks apart.

41. The method of any of embodiments 28-40 wherein the two time pointsare not more than four weeks apart, five weeks, or six weeks apart.

42. The method of any of embodiments 28-41, wherein the second timepoint is more than 1, 2, 3, 4, 5, 6, or 7 days before administration ofthe cell therapy.

43. The method of any of embodiments 28-42, wherein the cell therapycomprises cells engineered to express a recombinant receptor.

44. The method of any of embodiments 28-43, wherein the fold change inthe factor indicative of disease burden is associated with a risk ofdeveloping toxicity following administration of the cell therapy.

45. The method of any of embodiments 28-44, wherein the toxicity isneurotoxicity and/or cytokine release syndrome (CRS).

46. The method of any of embodiments 28-45, wherein the toxicity isearly toxicity that develops within 7 days of administration of the celltherapy.

47. The method of any of embodiments 28-46, wherein the toxicitydevelops within 3, 4, 5, 6, or 7 days of administration of the celltherapy.

48. The method of any of embodiments 28-47, wherein the toxicity is afirst sign of a fever or is a sustained fever following administrationof the cell therapy.

49. The method of any of embodiments 29-48, wherein the assessing of thefold change in a factor indicative of disease burden between two timepoints comprises a comparison to a threshold value, wherein thecomparison indicates the risk or likely risk of the subject developingtoxicity following administration of the cell therapy to the subject.

50. The method of any of embodiments 28-49, wherein the fold change inthe factor indicative of disease burden correlates to a risk that thesubject is or is likely to develop toxicity following administration ofthe cell therapy when it is administered.

51. The method of any of embodiments 28-50, wherein:

the subject is or is likely at risk of developing toxicity if the foldchange is at or above a threshold value; or

the subject is not or is likely not at risk of developing toxicity ifthe fold change is below a threshold value.

52. The method of embodiment 51, wherein the threshold value is a valuethat:

i) is within 25%, within 20%, within 15%, within 10%, or within 5% abovethe average fold change of the factor indicative of disease burdenand/or is within a standard deviation above the average fold change ofthe factor indicative of disease burden in a plurality of controlsubjects;

ii) is above the highest fold change of the factor indicative of diseaseburden, optionally within 50%, within 25%, within 20%, within 15%,within 10%, or within 5% above such highest fold change, measured in atleast one subject from among a plurality of control subjects; and/or

iii) is above the highest fold change as measured among more than 75%,80%, 85%, 90%, or 95%, or 98% of subjects from a plurality of controlsubjects.

53. The method of embodiment 52, wherein the plurality of controlsubjects are a group of subjects prior to receiving a cell therapy fortreating a disease or condition, said cell therapy containing cellsgenetically engineered with a recombinant receptor, wherein each of thesubjects of the group went on to develop toxicity, optionally earlytoxicity, optionally a fever or a sustained fever, within 7 days afterreceiving the cell therapy for treating the same disease or condition.

54. The method of any of embodiments 28-53, wherein if the assessingindicates the subject is or is likely to develop toxicity followingadministration of the cell therapy, the therapeutic regimen comprisesadministering to the subject:

-   -   i. an agent or other treatment capable of treating, preventing,        delaying, reducing or attenuating the development or risk of        development of a toxicity and the cell therapy, wherein        administration of the agent is to be administered (i) prior        to, (ii) within one, two, or three days of, (iii) concurrently        with and/or (iv) at first fever following, the initiation of        administration of the cell therapy to the subject;    -   ii. the cell therapy at a reduced dose or at a dose that is not        associated with risk of developing toxicity or severe toxicity,        or is not associated with a risk of developing a toxicity or        severe toxicity in a majority of subjects, and/or a majority of        subjects having a disease or condition that the subject has or        is suspected of having, following administration of the cell        therapy; and/or    -   iii. the cell therapy in an in-patient setting and/or with        admission to the hospital for one or more days, optionally        wherein the cell therapy is otherwise to be administered to        subjects on an outpatient basis or without admission to the        hospital for one or more days; or iv. an alternative therapeutic        treatment other than the cell therapy.

55. The method of any of embodiments 28-50, wherein if the assessingindicates the subject is not or is likely not to develop toxicityfollowing administration of the cell therapy, the therapeutic regimencomprises administering to the subject:

-   -   i. the cell therapy, optionally at a non-reduced dose,        optionally on an outpatient basis or without admission to the        hospital for one or more days;    -   ii. the cell therapy, wherein administration of the cell therapy        does not comprise administering, prior to or concurrently with        administering the cell therapy and/or prior to the development        of a sign or symptom of toxicity other than fever, an agent or        treatment capable of treating, preventing, delaying, or        attenuating the development of the toxicity; or    -   iii. the cell therapy in an outpatient setting and/or without        admission of the subject to the hospital overnight or for one or        more consecutive days and/or is without admission of the subject        to the hospital for one or more days.

56. The method of any of embodiments 28-50 wherein if the assessingindicates the subject is or is likely to develop toxicity followingadministration of the cell therapy, the cell therapy is not administeredto the subject.

57. The method of any of embodiments 1-56, wherein the disease orcondition is a cancer.

58. The method of embodiment 57, wherein the cancer is a myeloma,lymphoma or leukemia.

59. The method of any of embodiments 1-58, wherein the disease orcondition is a B cell malignancy.

60. The method of embodiment 59, wherein the B cell malignancy isselected from acute lymphoblastic leukemia (ALL), chronic lymphoblasticleukemia (CLL), acute myelogenous leukemia (AML), chronic myelogenousleukemia (CML), non-Hodgkin lymphoma (NHL), or Diffuse Large B-CellLymphoma (DLBCL), or a subtype of any of the foregoing.

61. The method of any of embodiments 27-60, wherein:

greater than or greater than about 30%, 35%, 40%, or 50% of the subjectstreated according to the method do not exhibit any grade of cytokinerelease syndrome (CRS) or neurotoxicity; and/or

at least at or about 45, 50, 60, 65, 70, 75, 80, 85, 90, 95% or about100% of subjects treated according to the method do not exhibit severeCRS, optionally grade 3 or higher, prolonged grade 3 or higher or grade4 or 5 CRS; and/or

at least at or about 45, 50, 60, 65, 70, 75, 80, 85, 90, 95% or about100% of subjects treated according to the method do not exhibit severeneurotoxicity, optionally grade 3 or higher, prolonged grade 3 or higheror grade 4 or 5 neurotoxicity; and/or

at least at or about 45, 50, 60, 65, 70, 75, 80, 85, 90, 95% or about100% of subjects treated according to the method do not exhibit cerebraledema.

62. The method of any of embodiments 27-61, wherein:

prior to initiation of administration of the dose of cells, the subjecthas not been administered an agent or treatment capable of treating,preventing, delaying, reducing or attenuating the development or risk ofdevelopment of a toxicity; and/or

the subject is not administered an agent or treatment for the treatmentor prevention or reduction or attenuation of a neurotoxicity and/or acytokine release syndrome or risk thereof, within a period of timefollowing administration of the dose, which period of time is optionallyat or about 1, 2, 3, 4, 5 days or is optionally at or about 6, 7, 8, 9,10, 11 days or is optionally 1 or 2 or 3 or 4 weeks; and/or

the subject is not administered an agent or treatment for the treatmentor prevention or reduction or attenuation of a neurotoxicity and/or acytokine release syndrome or risk thereof, following administration ofthe dose, prior to or unless the subject exhibits a sign or symptom ofthe toxicity and/or prior to or unless the subject exhibits a sign orsymptom of the toxicity other than a fever, optionally wherein the feveris not a sustained fever or the fever is or has been reduced or reducedby more than 1C after treatment with an antipyretic; and/or

the administration and any follow-up is carried out on an outpatientbasis and/or without admitting the subject to a hospital and/or withoutan overnight stay at a hospital and/or without requiring admission to oran overnight stay at a hospital, optionally unless or until the subjectexhibits a sustained fever or a fever that is or has not been reduced ornot reduced by more than 1° C. after treatment with an antipyretic.

63. The method of any of embodiments 27-62, wherein:

prior to initiation of administration of the dose of cells, the subjecthas not been administered an anti-IL-6 or anti-IL-6R antibody,optionally tocilizumab or siltuximab, and/or has not been administered asteroid, optionally dexamethasone

the subject is not administered an anti-IL-6 or anti-IL-6R antibody,optionally tocilizumab or siltuximab, and/or has not been administered asteroid, optionally dexamethasone, within a period of time followingadministration of the dose, which period of time is optionally at orabout 1, 2, 3, 4, 5 days or is optionally at or about 6, 7, 8, 9, 10, 11days or is optionally 1 or 2 or 3 or 4 weeks; and/or

the subject is not administered an anti-IL-6 or anti-IL-6R antibody,optionally tocilizumab or siltuximab, and/or has not been administered asteroid, optionally dexamethasone, following administration of the celldose, prior to, or unless, the subject exhibits a sign or symptom of atoxicity, optionally a neurotoxicity or CRS, and/or prior to, or unless,the subject exhibits a sign or symptom of a toxicity, optionally aneurotoxicity or CRS, other than a fever, optionally wherein the feveris not a sustained fever or the fever is or has been reduced or reducedby more than 1C after treatment with an antipyretic; and/or theadministration and any follow-up is carried out on an outpatient basisand/or without admitting the subject to a hospital and/or without anovernight stay at a hospital and/or without requiring admission to or anovernight stay at a hospital, optionally unless or until the subjectexhibits a sustained fever or a fever that is or has not been reduced ornot reduced by more than 1C after treatment with an antipyretic.

64. The method of any of embodiments 27-63, wherein:

the administration is carried out on an outpatient basis and/or withoutrequiring admission to or an overnight stay at a hospital; and

if the subject exhibits a sustained fever or a fever that is or has notbeen reduced or not reduced by more than 1C after treatment with anantipyretic, the subject is admitted to the hospital or to an overnightstay at a hospital and/or is administered an agent or treatment for thetreatment or prevention or reduction or attenuation of a neurotoxicityand/or a cytokine release syndrome or risk thereof.

65. The method of any of embodiments 1-64, wherein the cell therapy isor comprises tumor infiltrating lymphocytic (TIL) therapy.

66. The method of any of embodiments 1-64, wherein the cell therapycomprises cells engineered to express a recombinant receptor thatspecifically binds to an antigen associated with a disease or conditionand/or expressed in cells associated with the disease or condition.

67. The method of any of embodiments 1-66, wherein the cell therapy isan adoptive cell therapy.

68. The method of embodiment 66 or embodiment 67, wherein the cellscomprise immune cells.

69. The method of embodiment 68, wherein the immune cells are orcomprise T cells or NK cells.

70. The method of embodiment 69, wherein the immune cells are orcomprise T cells and the T cells comprise CD4+ and/or CD8+ T cells.

71. The method of any of embodiments 1-70, wherein the cell therapy is aT cell therapy comprising genetically engineered cells expressing arecombinant receptor.

72. The method of embodiment 71, wherein the recombinant receptor is a Tcell receptor or a functional non-T cell receptor.

73. The method of embodiment 71 or embodiment 72, wherein therecombinant receptor specifically binds to an antigen associated with adisease or condition and/or expressed in cells associated with thedisease or condition.

74. The method of embodiment 73, wherein the antigen is selected fromamong Trophoblast glycoprotein (TPBG also known as 5T4), 8H9, αvβ6integrin (avb6 integrin), B7-H3, B7-H6, B cell maturation antigen(BCMA), carbonic anhydrase 9 (CA9, also known as CAIX or G250), acancer-testes antigen, carbonic anhydrase 9 (CAIX), C-C Motif ChemokineLigand 1 (CCL-1), CD19, CD20, CD22, hepatitis B surface antigen, CD23,CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD123, CD133, CD138,CD171, carcinoembryonic antigen (CEA), a cyclin, cyclin A2, c-Met, dualantigen, chondroitin sulfate proteoglycan 4 (CSPG4), epidermal growthfactor protein (EGFR), epithelial glycoprotein 2 (EPG-2), epithelialglycoprotein 40 (EPG-40), ephrinB2, ephrin receptor A2 (EPHa2),ephrinB2, type III epidermal growth factor receptor mutation (EGFRvIII), estrogen receptor, folate receptor alpha, folate binding protein(FBP), Fc receptor like 5 (FCRL5; also known as Fc receptor homolog 5 orFCRH5), fetal acetylcholine receptor (Fetal AchR), G250/CAIX,ganglioside GD2, ganglioside GD3, glycoprotein 100 (gp100), glypican-3(GPC3), G Protein Coupled Receptor 5D (GPRC5D), Her2/neu (receptortyrosine kinase erbB2), Her3 (erb-B3), Her4 (erb-B4), erbB dimers, Humanhigh molecular weight-melanoma-associated antigen (HMW-MAA), Humanleukocyte antigen A1 (HLA-A1), Human leukocyte antigen A2 (HLA-A2),IL-22 receptor alpha (IL-22R-alpha), IL-13 receptor alpha 2 (IL-13Ra2),kinase insert domain receptor (kdr), kappa light chain, Leucine RichRepeat Containing 8 Family Member A (LRRC8A), Lewis Y, L1-cell adhesionmolecule (L1-CAM), CE7 epitope of L-CAM, Melanoma-associated antigen(MAGE)-A1, MAGE-A3, MAGE-A6, MAGE-A10, melan A (MART-1), mesothelin(MSLN), murine cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, neural celladhesion molecule (NCAM), natural killer group 2 member D (NKG2D), NKG2Dligands, cancer/testis antigen 1B (CTAG, also known as NY-ESO-1 andLAGE-2), O-acetylated GD2 (OGD2), oncofetal antigen, Preferentiallyexpressed antigen of melanoma (PRAME), a prostate specific antigen,prostate stem cell antigen (PSCA), progesterone receptor, survivin,prostate specific membrane antigen (PSMA), Receptor Tyrosine Kinase LikeOrphan Receptor 1 (ROR1), tumor-associated glycoprotein 72 (TAG72),Tyrosinase related protein 1 (TRP1, also known as TYRP1 or gp75),Tyrosinase related protein 2 (TRP2, also known as dopachrometautomerase, dopachrome delta-isomerase or DCT), vascular endothelialgrowth factor receptor (VEGF receptors; VEGFR), vascular endothelialgrowth factor receptor 2 (VEGF-R2), Wilms Tumor 1 (WT-1), apathogen-specific antigen.

75. The method of embodiment 73 or embodiment 74, wherein the CARcomprises an extracellular antigen-recognition domain that specificallybinds to the antigen and an intracellular signaling domain comprising anITAM, wherein optionally, the intracellular signaling domain comprisesan intracellular domain of a CD3-zeta (CD3ζ) chain; and/or wherein theCAR further comprises a costimulatory signaling region, which optionallycomprises a signaling domain of CD28 or 4-1BB.

76. The method of any of embodiments 71-75, wherein the geneticallyengineered cells comprise T cells or NK cells.

77. The method of any of embodiments 71-76, wherein the geneticallyengineered cells comprise T cells, and the T cells comprise CD4+ and/orCD8+ T cells.

78. The method of embodiment 77, wherein the T cells are primary T cellsobtained from a subject.

79. The method of any of embodiments 1-78, wherein the cells of the celltherapy are autologous to the subject or the cells are allogeneic to thesubject.

80. The method of any of embodiments 21, 22, 49, 51, or 52, wherein thethreshold fold change for SPD is about 5 fold, 6 fold, 7 fold, 8 fold,or 9 fold.

81. The method of any of embodiments 1-80, wherein the cell therapycomprises the administration of from or from about 1×10⁵ to 1×10⁸ totalrecombinant receptor-expressing cells, total T cells, or totalperipheral blood mononuclear cells (PBMCs), from or from about 5×10⁵ to1×10⁷ total recombinant receptor-expressing cells, total T cells, ortotal peripheral blood mononuclear cells (PBMCs) or from or from about1×10⁶ to 1×10⁷ total recombinant receptor-expressing cells, total Tcells, or total peripheral blood mononuclear cells (PBMCs), eachinclusive.

82. The method of any of embodiments 1-81, wherein the cell therapycomprises the administration of no more than 1×10⁸ total recombinantreceptor-expressing cells, total T cells, or total peripheral bloodmononuclear cells (PBMCs), no more than 1×10⁷ total recombinantreceptor-expressing cells, total T cells, or total peripheral bloodmononuclear cells (PBMCs), no more than 0.5×10⁷ total recombinantreceptor-expressing cells, total T cells, or total peripheral bloodmononuclear cells (PBMCs), no more than 1×10⁶ total recombinantreceptor-expressing cells, total T cells, or total peripheral bloodmononuclear cells (PBMCs), no more than 0.5×10⁶ total recombinantreceptor-expressing cells, total T cells, or total peripheral bloodmononuclear cells (PBMCs).

83. A kit, comprising (a) a cell therapy, said cell therapy comprisingcells genetically engineered with a recombinant receptor; and (b)instructions for administering the cell therapy to a subject, whereinthe instructions specify:

(1) assessing one or more factors indicative of tumor burden in asubject that is a candidate for treatment with the cell therapy, whereinthe assessing is performed at two time points prior to the subjectreceiving the cell therapy, and the factor indicative of tumor burden isa volumetric measure of the tumor or is an inflammatory marker in asample from a subject; and

(2) administering the cell therapy to the subject based on the foldchange in the factor indicative of disease burden between the two timepoints, wherein the fold change indicates the risk or likely risk of thesubject developing toxicity following administration of the cell therapyto the subject.

84. A kit, comprising (a) a cell therapy, said cell therapy comprisingcells genetically engineered with a recombinant receptor; and (b) one ormore reagents for assaying one or more factor indicative of diseaseburden, wherein the factor indicative of disease burden is aninflammatory marker selected from C-reactive protein (CRP), erythrocytesedimentation rate (ESR), albumin, ferritin, β2 microglobulin (β2-M), orlactate dehydrogenase (LDH).

85. The kit of embodiment 84, wherein the kit further comprisesinstructions for assessing the presence, level or amount at least one ofthe inflammatory marker in two or more samples obtained at two timepoints from a subject that is a candidate for treatment with the celltherapy and determining the fold-change in the factor between the twotime points, wherein the fold change indicates the risk or likely riskof the subject developing toxicity following administration of the celltherapy to the subject.

86. The kit of embodiment 85, wherein the kit further comprisesinstructions for administering a therapeutic regimen comprising the celltherapy to the subject following or based on the fold change of the atleast one inflammatory marker.

87. A kit, comprising (a) a cell therapy, said cell therapy comprisingcells genetically engineered with a recombinant receptor; and (b)instructions for administering a therapeutic regimen comprising the celltherapy following or based on the results of assessing the subject for afold change in a factor indicative of disease burden between two timepoints prior to receiving a cell therapy, wherein the subject is acandidate for treatment with the cell therapy.

88. The kit of embodiment 87, wherein the factor indicative of diseaseburden is a volumetric measure of a tumor or is an inflammatory markerin a sample from a subject.

89. The kit of any of embodiments 83, 87, or 88, wherein the factorindicative of disease burden is a volumetric measure and the volumetricmeasure is a sum of the products of diameters (SPD), longest tumordiameters (LD), sum of longest tumor diameters (SLD), necrosis, tumorvolume, necrosis volume, necrosis-tumor ratio (NTR), peritumoral edema(PTE), and edema-tumor ratio (ETR).

90. The kit of any of embodiments 83, or 87-89, wherein the volumetricmeasure is measured using computed tomography (CT), positron emissiontomography (PET), and/or magnetic resonance imaging (MRI) of thesubject.

91. The kit of any of embodiments 83-88, wherein the kit comprisesreagents for detecting C-reactive protein (CRP), erythrocytesedimentation rate (ESR), albumin, ferritin, 32 microglobulin (β2-M), orlactate dehydrogenase (LDH).

92. The kit of any of embodiments 83-88 or 91, wherein the inflammatorymarker is assessed using a colorimetric assay or an immunoassay.

93. The kit of any of embodiments 83-88 or 91-92, wherein theinflammatory marker is assessed using an immunoassay and the immunoassayis selected from enzyme-linked immunosorbent assay (ELISA), enzymeimmunoassay (EIA), radioimmunoassay (RIA), surface plasmon resonance(SPR), Western Blot, Lateral flow assay, immunohistochemistry, proteinarray or immuno-PCR (iPCR).

94. The kit of any of embodiments 83-88 or 91-93, wherein the sample isor comprises a blood sample, plasma sample, or serum sample.

95. The kit of any of embodiments 83-94, wherein the subject is a human.

96. The kit of any of embodiments 83-95, wherein the two time pointscomprises a first time point and a second time point, and wherein thefold change is a ratio of the factor indicative of disease burden at thea first time point and a second time point.

97. The kit of any of embodiments 83-96, wherein the two time points areboth no more than one month or two months prior to receiving the celltherapy.

98. The kit of any of embodiments 83-97 wherein the two time points arenot less than one week, two weeks, three weeks, four weeks, or fiveweeks apart.

99. The kit of any of embodiments 83-98, wherein the two time points arenot less than three weeks apart.

100. The kit of any of embodiments 83-99 wherein the two time points arenot more than four weeks apart, five weeks, or six weeks apart.

101. The kit of any of embodiments 83-100, wherein the second time pointis more than 1, 2, 3, 4, 5, 6, or 7 days before administration of thecell therapy.

102. The kit of any of embodiments 83-101, wherein:

the instructions specify that the fold change in the factor indicativeof disease burden indicates the subject is or is likely at risk ofdeveloping toxicity if the fold change is at or above a threshold value;or

the instructions specify that the fold change in the factor indicativeof disease burden indicates the subject is not or is likely not at riskof developing toxicity if the fold change is below a threshold value.

103. The kit of embodiment 102, wherein the threshold value is a valuethat:

i) is within 25%, within 20%, within 15%, within 10%, or within 5% abovethe average fold change of the factor indicative of disease burdenand/or is within a standard deviation above the average fold change ofthe factor indicative of disease burden in a plurality of controlsubjects;

ii) is above the highest fold change of the factor indicative of diseaseburden, optionally within 50%, within 25%, within 20%, within 15%,within 10%, or within 5% above such highest fold change, measured in atleast one subject from among a plurality of control subjects; and/or

iii) is above the highest fold change as measured among more than 75%,80%, 85%, 90%, or 95%, or 98% of subjects from a plurality of controlsubjects.

104. The kit of embodiment 103, wherein the plurality of controlsubjects are a group of subjects prior to receiving a cell therapy fortreating a disease or condition, said cell therapy containing cellsgenetically engineered with a recombinant receptor, wherein each of thesubjects of the group went on to develop toxicity, optionally earlytoxicity, optionally a fever or a sustained fever, within 7 days afterreceiving the cell therapy for treating the same disease or condition.

105. The kit of any of embodiments 87-104, wherein the instructionsspecify if the fold change indicates the subject is or is likely todevelop toxicity, selecting the subject for administration of atherapeutic regimen, the therapeutic regimen comprising administering tothe subject:

i. an agent or other treatment capable of treating, preventing,delaying, reducing or attenuating the development or risk of developmentof a toxicity and the cell therapy, wherein administration of the agentis to be administered (i) prior to, (ii) within one, two, or three daysof, (iii) concurrently with and/or (iv) at first fever following, theinitiation of administration of the cell therapy to the subject;

ii. the cell therapy at a reduced dose or at a dose that is notassociated with risk of developing toxicity or severe toxicity, or isnot associated with a risk of developing a toxicity or severe toxicityin a majority of subjects, and/or a majority of subjects having adisease or condition that the subject has or is suspected of having,following administration of the cell therapy; and/or

iii. the cell therapy in an in-patient setting and/or with admission tothe hospital for one or more days, optionally wherein the cell therapyis otherwise to be administered to subjects on an outpatient basis orwithout admission to the hospital for one or more days; or iv. analternative therapeutic treatment other than the cell therapy.

106. The kit of any of embodiments 87-104, wherein the instructionsspecify if the fold change indicates the subject is not or is likely notat risk of developing toxicity, selecting the subject for administrationof a therapeutic regimen, the therapeutic regimen comprisingadministering to the subject:

i. the cell therapy, optionally at a non-reduced dose, optionally on anoutpatient basis or without admission to the hospital for one or moredays;

ii. the cell therapy, wherein administration of the cell therapy doesnot comprise administering, prior to or concurrently with administeringthe cell therapy and/or prior to the development of a sign or symptom oftoxicity other than fever, an agent or treatment capable of treating,preventing, delaying, or attenuating the development of the toxicity; or

iii. the cell therapy in an outpatient setting and/or without admissionof the subject to the hospital overnight or for one or more consecutivedays and/or is without admission of the subject to the hospital for oneor more days.

107. The kit of any of embodiments 87-104, wherein the instructionsspecify if the fold change indicates the subject is or is likely todevelop toxicity, the therapeutic regimen is not administered to thesubject.

108. The kit of embodiment 106, wherein the instructions specify:

the administration is carried out on an outpatient basis and/or withoutrequiring admission to or an overnight stay at a hospital; and

if the subject exhibits a sustained fever or a fever that is or has notbeen reduced or not reduced by more than 1C after treatment with anantipyretic, the subject is admitted to the hospital or to an overnightstay at a hospital and/or is administered an agent or treatment for thetreatment or prevention or reduction or attenuation of a neurotoxicityand/or a cytokine release syndrome or risk thereof.

109. The kit of any of embodiments 83-108, wherein the toxicity isneurotoxicity and/or cytokine release syndrome (CRS).

110. The kit of any of embodiments 87-109, wherein the toxicity is earlytoxicity that develops within 7 days of administration of the celltherapy.

111. The kit of any of embodiments 87-110, wherein the toxicity developswithin 3, 4, 5, 6, or 7 days of administration of the cell therapy.

112. The kit of any of embodiments 87-111, wherein the toxicity is afirst sign of a fever or is a sustained fever following administrationof the cell therapy.

113. The kit of any of embodiments 83-112, wherein the cell therapy isor comprises tumor infiltrating lymphocytic (TIL) therapy.

114. The kit of any of embodiments 83-113, wherein the cell therapycomprises cells engineered to express a recombinant receptor thatspecifically binds to an antigen associated with a disease or conditionand/or expressed in cells associated with the disease or condition.

115. The kit of any of embodiments 83-114, wherein the cell therapy isan adoptive cell therapy.

116. The kit of embodiment 114 or embodiment 115, wherein the cellscomprise immune cells.

117. The kit of embodiment 116, wherein the immune cells are or compriseT cells or NK cells.

118. The kit of embodiment 117, wherein the immune cells are or compriseT cells and the T cells comprise CD4+ and/or CD8+ T cells.

119. The kit of any of embodiments 83-118, wherein the cell therapy is aT cell therapy comprising genetically engineered cells expressing arecombinant receptor.

120. The kit of embodiment 104, wherein the disease or condition is acancer.

121. The kit of embodiment 104 or embodiment 120, wherein the cancer isa myeloma, leukemia or lymphoma.

122. The kit of embodiment 104, wherein the disease or condition is a Bcell malignancy.

123. The kit of embodiment 122, wherein the B cell malignancy isselected from acute lymphoblastic leukemia (ALL), chronic lymphoblasticleukemia (CLL), acute myelogenous leukemia (AML), chronic myelogenousleukemia (CML), non-Hodgkin lymphoma (NHL), or Diffuse Large B-CellLymphoma (DLBCL), or a subtype of any of the foregoing.

124. The kit of any of embodiments 114-123, wherein the antigen isselected from among Trophoblast glycoprotein (TPBG also known as 5T4),8H9, αvβ6 integrin (avb6 integrin), B7-H3, B7-H6, B cell maturationantigen (BCMA), carbonic anhydrase 9 (CA9, also known as CAIX or G250),a cancer-testes antigen, carbonic anhydrase 9 (CAIX), C-C MotifChemokine Ligand 1 (CCL-1), CD19, CD20, CD22, hepatitis B surfaceantigen, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD123,CD133, CD138, CD171, carcinoembryonic antigen (CEA), a cyclin, cyclinA2, c-Met, dual antigen, chondroitin sulfate proteoglycan 4 (CSPG4),epidermal growth factor protein (EGFR), epithelial glycoprotein 2(EPG-2), epithelial glycoprotein 40 (EPG-40), ephrinB2, ephrin receptorA2 (EPHa2), ephrinB2, type III epidermal growth factor receptor mutation(EGFR vIII), estrogen receptor, folate receptor alpha, folate bindingprotein (FBP), Fc receptor like 5 (FCRL5; also known as Fc receptorhomolog 5 or FCRH5), fetal acetylcholine receptor (Fetal AchR),G250/CAIX, ganglioside GD2, ganglioside GD3, glycoprotein 100 (gp100),glypican-3 (GPC3), G Protein Coupled Receptor 5D (GPRC5D), Her2/neu(receptor tyrosine kinase erbB2), Her3 (erb-B3), Her4 (erb-B4), erbBdimers, Human high molecular weight-melanoma-associated antigen(HMW-MAA), Human leukocyte antigen A1 (HLA-A1), Human leukocyte antigenA2 (HLA-A2), IL-22 receptor alpha (IL-22R-alpha), IL-13 receptor alpha 2(IL-13Ra2), kinase insert domain receptor (kdr), kappa light chain,Leucine Rich Repeat Containing 8 Family Member A (LRRC8A), Lewis Y,L1-cell adhesion molecule (L1-CAM), CE7 epitope of L-CAM,Melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MAGE-A10, melanA (MART-1), mesothelin (MSLN), murine cytomegalovirus (CMV), mucin 1(MUC1), MUC16, neural cell adhesion molecule (NCAM), natural killergroup 2 member D (NKG2D), NKG2D ligands, cancer/testis antigen 1B (CTAG,also known as NY-ESO-1 and LAGE-2), O-acetylated GD2 (OGD2), oncofetalantigen, Preferentially expressed antigen of melanoma (PRAME), aprostate specific antigen, prostate stem cell antigen (PSCA),progesterone receptor, survivin, prostate specific membrane antigen(PSMA), Receptor Tyrosine Kinase Like Orphan Receptor 1 (ROR1),tumor-associated glycoprotein 72 (TAG72), Tyrosinase related protein 1(TRP1, also known as TYRP1 or gp75), Tyrosinase related protein 2 (TRP2,also known as dopachrome tautomerase, dopachrome delta-isomerase orDCT), vascular endothelial growth factor receptor (VEGF receptors;VEGFR), vascular endothelial growth factor receptor 2 (VEGF-R2), WilmsTumor 1 (WT-1), a pathogen-specific antigen.

125. The kit of any of embodiments 104-124, wherein the recombinantreceptor is a T cell receptor or a functional non-T cell receptor.

126. The kit of any of embodiments 104-125, wherein the recombinantreceptor is a chimeric antigen receptor (CAR).

127. The kit of embodiment 126, wherein the CAR comprises anextracellular antigen-recognition domain that specifically binds to theantigen and an intracellular signaling domain comprising an ITAM,wherein optionally, the intracellular signaling domain comprises anintracellular domain of a CD3-zeta (CD3ζ) chain; and/or wherein the CARfurther comprises a costimulatory signaling region, which optionallycomprises a signaling domain of CD28 or 4-1BB.

128. The kit of any of embodiments 83-127, further comprising an agentor other treatment capable of treating, preventing, delaying, reducingor attenuating the development or risk of development of a toxicity.

129. The kit of embodiment 128, wherein the agent or other treatment isor comprises one or more of a steroid; an antagonist or inhibitor of acytokine receptor or cytokine selected from among IL-10, IL-10R, IL-6,IL-6 receptor, IFN7, IFNGR, IL-2, IL-2R/CD25, MCP-1, CCR2, CCR4, MIP10,CCR5, TNFalpha, TNFR1, IL-1, and IL-1Ralpha/IL-1beta; or an agentcapable of preventing, blocking or reducing microglial cell activity orfunction.

130. The kit of embodiment 129, wherein the antagonist or inhibitor isor comprises an agent selected from among an antibody or antigen-bindingfragment, a small molecule, a protein or peptide and a nucleic acid.

131. The kit of any of embodiments 128-130, wherein the agent or othertreatment is an anti-IL-6 antibody or an anti-IL6 receptor antibody.

132. The kit of any of embodiments 128-131, wherein the agent or othertreatment is or comprises an agent selected from among tocilizumab,siltuximab, clazakizumab, sarilumab, olokizumab (CDP6038), elsilimomab,ALD518/BMS-945429, sirukumab (CNTO 136), CPSI-2634, ARGX-109, FE301 andFM101.

133. The kit of any of embodiments 128-132, wherein the agent or othertreatment is or comprises tocilizumab.

134. The kit of any of embodiments 128-133, wherein the agent or othertreatment is or comprises siltuximab.

135. The kit of any of embodiments 129-134, wherein the steroid is orcomprises dexamethasone.

136. The kit of any of embodiments 104-135, wherein the geneticallyengineered cells comprise T cells, and the T cells comprise CD4+ and/orCD8+ T cells.

137. The kit of embodiment 136, wherein the T cells are primary T cellsobtained from a subject.

138. The kit of any of embodiments 83-137, wherein the cells of the celltherapy are autologous to the subject.

139. The kit of any of embodiments 83-138, wherein the cells areallogeneic to the subject.

140. The kit of embodiment 102 or embodiment 103, wherein the thresholdvalue for SPD is about 5 fold, 6 fold, 7 fold, 8 fold, or 9 fold.

VIII. EXAMPLES

The following examples are included for illustrative purposes only andare not intended to limit the scope of the invention.

Example 1: Administration of an Anti-CD19 CAR-Expressing Cells andAssessment of Tumor Burden and Toxicity in Subjects with Relapsed andRefractory Non-Hodgkin's Lymphoma

Therapeutic CAR+ T cell compositions containing autologous T cellsexpressing a chimeric antigen-receptor (CAR) specific for CD19 wereadministered to adult human subjects with relapsed or refractory (R/R)aggressive non-Hodgkin's lymphoma (NHL) after failure of at least 2lines of therapy. The therapeutic T cell compositions administered hadbeen generated by a process including immunoaffinity-based enrichment ofCD4+ and CD8+ cells from leukapheresis samples from the individualsubjects to be treated. Isolated CD4+ and CD8+ T cells were activatedand transduced with a viral vector encoding an anti-CD19 CAR, followedby expansion and cryopreservation of the engineered cell populations.The CAR contained an anti-CD19 scFv derived from a murine antibody, animmunoglobulin-derived spacer, a transmembrane domain derived from CD28,a costimulatory region derived from 4-1BB, and a CD3-zeta intracellularsignaling domain.

The cryopreserved cell compositions were thawed prior to intravenousadministration. The therapeutic T cell dose was administered as adefined cell composition by administering a formulated CD4+CAR+ cellpopulation and a formulated CD8+CAR+ population administered at a targetratio of approximately 1:1. Subjects were administered a single dosecontaining 5×10⁷ total CAR-expressing T cells (via separate infusions ofCD4+ CAR-expressing T cells and CD8+ CAR-expressing T cells). Beginningat three (3) days prior to CAR+ T cell infusion, subjects received alymphodepleting chemotherapy.

To obtain a volumetric measure of tumor burden, PET scans were obtainedat two time points including at screening and prior to administration oflymphodepletion treatment to confirm PET-avid disease. The sums of theperpendicular diameters (SPDs) was determined for measurable lesions onPET scan. The SPD at screening scan and SPD measured immediately priorto administration of the lymphodepletion treatment (pre-lymphodepletingtreatment SPD) was compared, and the fold change in SPD over the periodbetween these two timepoints determined for a number of the subjects.Ferritin levels in serum were similarly obtained just prior toadministration of the lymphodepleting chemotherapy (Pre-lymphodepletingtreatment Ferritin). Subjects were monitored and assessed for variousoutcomes, including the presence or absence of symptoms of outcomesincluding outcomes indicative of toxicity (such as CRS or neurotoxicity(NT).

Table E1 summarizes results for a study comparing levels of the measureof tumor burden (SPD) at the two time points and incidence of early CRS.Among ten of the subjects treated as described above for which SPDmeasurements and fold change had been determined, early toxicity (CRS).within the first 7 days following initiation of administration of thecell therapy was observed in three subjects ( 3/10) and no signs of CRSor neurotoxicity was observed within the first 3 weeks afteradministration of the cell therapy in seven subjects ( 7/10). Acorrelation between the relative change in SPD and early toxicity wasobserved.

Pre-lym- Median phodepleting Ferritin Pre-lym- treatment Among SubjectScreening phodepleting Fold Ferritin Group of # SPD treatment SPD change(ng/mL) Subjects Group of Subjects Exhibiting Early Toxicity 1 19.6 26813.7 4399 1224 2 4.2 79 18.8 172 3 19 245 12.9 1224 Group of SubjectsNot Exhibiting Early Toxicity 4 8 12 1.5 514 395 5 6 7 1.2 395 6 9.7 959.8 1166 7 4.2 16 3.8 236 9 4.8 15 3.1 960 10 8.3 6.9 0.83 262 11 6.1 71.1 20

The present invention is not intended to be limited in scope to theparticular disclosed embodiments, which are provided, for example, toillustrate various aspects of the invention. Various modifications tothe compositions and methods described will become apparent from thedescription and teachings herein. Such variations may be practicedwithout departing from the true scope and spirit of the disclosure andare intended to fall within the scope of the present disclosure.

SEQUENCES SEQ ID NO. SEQUENCE DESCRIPTION  1 ESKYGPPCPPCP spacer(IgG4hinge) (aa) Homo sapiens  2 GAATCTAAGTACGGACCGCCCTGCCCCCCTTGCCCTspacer (IgG4hinge) (nt) homo sapiens  3ESKYGPPCPPCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGF Hinge-CH3YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSR spacerWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK Homo sapiens  4ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVV Hinge-CH2-VDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSV CH3 spacerLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQV Homo sapiensYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHN HYTQKSLSLSLGK  5RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEE IgD-hinge-FcKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRD Homo sapiensKATFTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVV SHEDSRTLLNASRSLEVSYVTDH  6LEGGGEGRGSLLTCGDVEENPGPR T2A artificial  7MLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIK tEGFRHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITG artificialFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGI GLFM  8FWVLVVVGGVLACYSLLVTVAFIIFWV CD28 (amino acids 153-179 of AccessionNo. P10747) Homo sapiens  9 IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVCD28 (amino VVGGVLACYSLLVTVAFIIFWV acids 114-179 of AccessionNo. P10747) Homo sapiens 10 RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSCD28 (amino acids 180-220 of P10747) Homo sapiens 11RSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS CD28 (LL to GG) Homo sapiens12 KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL 4-1BB (amino acids 214-255of Q07011.1) Homo sapiens 13 RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDCD3 zeta PEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK Homo sapiensGHDGLYQGLSTATKDTYDALHMQALPPR 14RVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRD CD3 zetaPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK Homo sapiensGHDGLYQGLSTATKDTYDALHMQALPPR 15RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRD CD3 zetaPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK Homo sapiensGHDGLYQGLSTATKDTYDALHMQALPPR 16RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGD tEGFRSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEI artificialIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPK IPSIATGMVGALLLLLVVALGIGLFM 17EGRGSLLTCGDVEENPGP T2A artificial 18 GSGATNFSLLKQAGDVEENPGP P2A 19ATNFSLLKQAGDVEENPGP P2A 20 QCTNYALLKLAGDVESNPGP E2A 21VKQTLNFDLLKLAGDVESNPGP F2A 22PGGG-(SGGGG)5-P- wherein P is proline, G is glycine and S is serineLinker 23 GSADDAKKDAAKKDGKS Linker 24 MALPVTALLLPLALLLHA CD8 alphasignal peptide 25atgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcctgatcccaGMCSFR alpha chain signal sequence 26 MLLLVTSLLLCELPHPAFLLIP GMCSFRalpha chain signal sequence 27Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Hinge 28ELKTPLGDTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCP Hinge EPKSCDTPPPCPRCP 29Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Hinge 30Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Hinge 31Tyr Gly Pro Pro Cys Pro Pro Cys Pro Hinge 32Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Hinge 33Glu Val Val Val Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Hinge 34QQGNTLPYT LC-CDR3 35 RASQDISKYLN CDR L1 36 SRLHSGV CDR L2 37 GNTLPYTFGCDR L3 38 DYGVS CDR H1 39 VIWGSETTYYNSALKS CDR H2 40 YAMDYWG CDR H3 41EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWL VHGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS 42DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLI VLYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYT FGGGTKLEIT 43DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLI scFvYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTS VTVSS 44 KASQNVGTNVACDR L1 45 SATYRNS CDR L2 46 QQYNRYPYT CDR L3 47 SYWMN CDR H1 48QIYPGDGDTNYNGKFKG CDR H2 49 KTISSVVDFYFDY CDR H3 50EVKLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLE VHWIGQIYPGDGDTNYNGKFKGQATLTADKSSSTAYMQLSGLTSEDSAVYFCARKTISSVVDFYFDYWGQGTTVTVSS 51DIELTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKPLI VLYSATYRNSGVPDRFTGSGSGTDFTLTITNVQSKDLADYFCQQYNRYPY TSGGGTKLEIKR 52GGGGSGGGGSGGGGS Linker 53 EVKLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEscFv WIGQIYPGDGDTNYNGKFKGQATLTADKSSSTAYMQLSGLTSEDSAVYFCARKTISSVVDFYFDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIELTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKPLIYSATYRNSGVPDRFTGSGSGTDFTLTITNVQSKDLADYFCQQYNRYPYTS GGGTKLEIKR 54HYYYGGSYAMDY HC-CDR3 55 HTSRLHS LC-CDR2 56 GSTSGSGKPGSGEGSTKG Linker 57gacatccagatgacccagaccacctccagcctgagcgccagcctgggcgaccgggtgaccatcagctgccgSequenceggccagccaggacatcagcaagtacctgaactggtatcagcagaagcccgacggcaccgtcaagctgctgatencodingctaccacaccagccggctgcacagcggcgtgcccagccggtttagcggcagcggctccggcaccgactacascFvgcctgaccatctccaacctggaacaggaagatatcgccacctacttttgccagcagggcaacacactgccctacacctttggcggcggaacaaagctggaaatcaccggcagcacctccggcagcggcaagcctggcagcggcgagggcagcaccaagggcgaggtgaagctgcaggaaagcggccctggcctggtggcccccagccagagcctgagcgtgacctgcaccgtgagcggcgtgagcctgcccgactacggcgtgagctggatccggcagccccccaggaagggcctggaatggctgggcgtgatctggggcagcgagaccacctactacaacagcgccctgaagagccggctgaccatcatcaaggacaacagcaagagccaggtgttcctgaagatgaacagcctgcagaccgacgacaccgccatctactactgcgccaagcactactactacggcggcagctacgccatggactactggggccagggcaccagcgtgaccgtgagcagc 58 X1PPX2P HingeX1 is glycine, cysteine or arginine X2 is cysteine or threonine 59Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Hinge 60MPLLLLLPLLWAGALA CD33 signal peptide

What is claimed:
 1. A method of assessing a risk of a toxicity or atoxicity-related outcome, following administration of a cell therapy,the method comprising: (1) assessing a factor indicative of diseaseburden at two time points, each prior to administration of a celltherapy, from a subject that is a candidate for receiving a cell therapyfor treatment of a disease or condition; and (2) determining a foldchange in the factor indicative of disease burden between the two timepoints, wherein the fold change indicates the risk or likely risk of thesubject developing a toxicity following administration of the therapy tothe subject.
 2. The method of claim 1, wherein the factor indicative ofdisease burden is a volumetric measure of a tumor or is an inflammatorymarker in a sample from a subject.
 3. The method of claim 2, wherein thefactor indicative of disease burden is a volumetric measure and thevolumetric measure is a sum of the products of diameters (SPD), longesttumor diameters (LD), sum of longest tumor diameters (SLD), tumorvolume, necrosis volume, necrosis-tumor ratio (NTR), peritumoral edema(PTE), and edema-tumor ratio (ETR).
 4. The method of claim 2 or claim 3,wherein the volumetric measure is assessed using computed tomography(CT), positron emission tomography (PET), and/or magnetic resonanceimaging (MRI) of the subject.
 5. The method of claim 2, wherein thefactor indicative of disease burden is an inflammatory marker and theinflammatory marker is C-reactive protein (CRP), erythrocytesedimentation rate (ESR), albumin, ferritin, β2 microglobulin (β2-M), orlactate dehydrogenase (LDH).
 6. The method of claim 2 or claim 5,wherein the sample is or comprises a blood sample, plasma sample, orserum sample.
 7. The method of any of claim 2, 5 or 6, wherein theinflammatory marker is assessed using a colorimetric assay or animmunoassay.
 8. The method of claim 7, wherein the inflammatory markeris assessed using an immunoassay and the immunoassay is selected fromenzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA),radioimmunoassay (RIA), surface plasmon resonance (SPR), Western Blot,Lateral flow assay, immunohistochemistry, protein array or immuno-PCR(iPCR).
 9. The method of any of claims 1-8, wherein the subject is ahuman.
 10. The method of any of claims 1-9, wherein the two time pointscomprises a first time point and a second time point, and wherein thefold change is a ratio of the factor indicative of disease burden at thefirst time point and the second time point.
 11. The method of any ofclaims 1-10, wherein the two time points are both no more than one monthor two months prior to receiving the cell therapy.
 12. The method of anyof claims 1-11, wherein the two time points are not less than one week,two weeks, three weeks, four weeks, or five weeks apart.
 13. The methodof any of claims 1-12, wherein the two time points are not less thanthree weeks apart.
 14. The method of any of claims 1-13, wherein the twotime points are not more than four weeks apart, five weeks, or six weeksapart.
 15. The method of any of claims 1-14, wherein the second timepoint is more than 1, 2, 3, 4, 5, 6, or 7 days before administration ofthe cell therapy.
 16. The method of any of claims 1-15, wherein the celltherapy comprises cells engineered to express a recombinant receptor.17. The method of any of claims 1-16, wherein the toxicity isneurotoxicity and/or cytokine release syndrome (CRS).
 18. The method ofany of claims 1-17, wherein the toxicity is early toxicity that developswithin 7 days of administration of the cell therapy.
 19. The method ofany of claims 1-18, wherein the toxicity develops within 3, 4, 5, 6, or7 days of administration of the cell therapy.
 20. The method of any ofclaims 1-19, wherein the toxicity is a first sign of a fever or is asustained fever following administration of the cell therapy.
 21. Themethod of any of claims 1-20, wherein: the subject is or is likely atrisk of developing toxicity if the fold change is at or above athreshold value; or the subject is not or is likely not at risk ofdeveloping toxicity if the fold change is below a threshold value. 22.The method of claim 21, wherein the threshold value is a value that: i)is within 25%, within 20%, within 15%, within 10%, or within 5% abovethe average fold change of the factor indicative of disease burdenand/or is within a standard deviation above the average fold change ofthe factor indicative of disease burden in a plurality of controlsubjects; ii) is above the highest fold change of the factor indicativeof disease burden, optionally within 50%, within 25%, within 20%, within15%, within 10%, or within 5% above such highest fold change, measuredin at least one subject from among a plurality of control subjects;and/or iii) is above the highest fold change as measured among more than75%, 80%, 85%, 90%, or 95%, or 98% of subjects from a plurality ofcontrol subjects.
 23. The method of claim 22, wherein the plurality ofcontrol subjects are a group of subjects prior to receiving a celltherapy for treating a disease or condition, said cell therapycontaining cells genetically engineered to express a recombinantreceptor, wherein each of the subjects of the group went on to developtoxicity, optionally early toxicity, optionally a fever or a sustainedfever, within 7 days after receiving the cell therapy for treating thesame disease or condition.
 24. The method of any of claim 1-23, whereinif the subject is indicated as likely to develop toxicity, selecting thesubject for administration of a therapeutic regimen, the therapeuticregimen comprising administering to the subject: i. an agent or othertreatment capable of treating, preventing, delaying, reducing orattenuating the development or risk of development of a toxicity and thecell therapy, wherein administration of the agent is to be administered(i) prior to, (ii) within one, two, or three days of, (iii) concurrentlywith and/or (iv) at first fever following, the initiation ofadministration of the cell therapy to the subject; ii. the cell therapyat a reduced dose or at a dose that is not associated with risk ofdeveloping toxicity or severe toxicity, or is not associated with a riskof developing a toxicity or severe toxicity in a majority of subjects,and/or a majority of subjects having a disease or condition that thesubject has or is suspected of having, following administration of thecell therapy; and/or iii. the cell therapy in an in-patient settingand/or with admission to the hospital for one or more days, optionallywherein the cell therapy is otherwise to be administered to subjects onan outpatient basis or without admission to the hospital for one or moredays; or iv. an alternative therapeutic treatment other than the celltherapy.
 25. The method of any of claims 1-23, wherein if subject isindicated as likely not at risk of developing toxicity, selecting thesubject for administration of a therapeutic regimen, the therapeuticregimen comprising administering to the subject: i. the cell therapy,optionally at a non-reduced dose, optionally on an outpatient basis orwithout admission to the hospital for one or more days; ii. the celltherapy, wherein administration of the cell therapy does not compriseadministering, prior to or concurrently with administering the celltherapy and/or prior to the development of a sign or symptom of toxicityother than fever, an agent or other treatment capable of treating,preventing, delaying, or attenuating the development of the toxicity; oriii. the cell therapy in an outpatient setting and/or without admissionof the subject to the hospital overnight or for one or more consecutivedays and/or is without admission of the subject to the hospital for oneor more days.
 26. The method of any of claims 1-23, wherein if thesubject is indicated as likely to develop toxicity followingadministration of the cell therapy, the cell therapy is not administeredto the subject.
 27. The method of claim 24 or claim 25, furthercomprising administering the therapeutic regimen to the selectedsubject.
 28. A method of treatment, the method comprising administeringa therapeutic regimen to a subject that is a candidate for receiving acell therapy for treatment of a disease or condition, wherein theadministration is carried out following or based on the results ofassessing the subject for a fold change in a factor indicative ofdisease burden between two time points prior to receiving a celltherapy.
 29. The method of claim 28, wherein the factor indicative ofdisease burden is a volumetric measure of a tumor or is an inflammatorymarker in a sample from a subject.
 30. The method of claim 29, whereinthe factor indicative of disease burden is a volumetric measure and thevolumetric measure is a sum of the products of diameters (SPD), longesttumor diameters (LD), sum of longest tumor diameters (SLD), necrosis,tumor volume, necrosis volume, necrosis-tumor ratio (NTR), peritumoraledema (PTE), and edema-tumor ratio (ETR).
 31. The method of claim 29 orclaim 30, wherein the volumetric measure is assessed using computedtomography (CT), positron emission tomography (PET), and/or magneticresonance imaging (MRI) of the subject.
 32. The method of claim 29,wherein the factor indicative of disease burden is an inflammatorymarker and the inflammatory marker is C-reactive protein (CRP),erythrocyte sedimentation rate (ESR), albumin, ferritin, β2microglobulin (β2-M), or lactate dehydrogenase (LDH).
 33. The method ofclaim 29 or claim 32, wherein the sample is or comprises a blood sample,plasma sample, or serum sample.
 34. The method of any of claim 29, 32 or33, wherein the inflammatory marker is assessed using a colorimetricassay or an immunoassay.
 35. The method of claim 34, wherein theinflammatory marker is assessed using an immunoassay and the immunoassayis selected from enzyme-linked immunosorbent assay (ELISA), enzymeimmunoassay (EIA), radioimmunoassay (RIA), surface plasmon resonance(SPR), Western Blot, Lateral flow assay, immunohistochemistry, proteinarray or immuno-PCR (iPCR).
 36. The method of any of claims 28-35,wherein the subject is a human.
 37. The method of any of claims 28-36,wherein the two time points comprises a first time point and a secondtime point, and wherein the fold change is a ratio of the factorindicative of disease burden at the first time point and the second timepoint.
 38. The method of any of claims 28-37, wherein the two timepoints are both no more than one month or two months prior to receivingthe cell therapy.
 39. The method of any of claims 28-38, wherein the twotime points are not less than one week, two weeks, three weeks, fourweeks, or five weeks apart.
 40. The method of any of claims 28-39,wherein the two time points are not less than three weeks apart.
 41. Themethod of any of claims 28-40 wherein the two time points are not morethan four weeks apart, five weeks, or six weeks apart.
 42. The method ofany of claims 28-41, wherein the second time point is more than 1, 2, 3,4, 5, 6, or 7 days before administration of the cell therapy.
 43. Themethod of any of claims 28-42, wherein the cell therapy comprises cellsengineered to express a recombinant receptor.
 44. The method of any ofclaims 28-43, wherein the fold change in the factor indicative ofdisease burden is associated with a risk of developing toxicityfollowing administration of the cell therapy.
 45. The method of any ofclaims 28-44, wherein the toxicity is neurotoxicity and/or cytokinerelease syndrome (CRS).
 46. The method of any of claims 28-45, whereinthe toxicity is early toxicity that develops within 7 days ofadministration of the cell therapy.
 47. The method of any of claims28-46, wherein the toxicity develops within 3, 4, 5, 6, or 7 days ofadministration of the cell therapy.
 48. The method of any of claims28-47, wherein the toxicity is a first sign of a fever or is a sustainedfever following administration of the cell therapy.
 49. The method ofany of claims 29-48, wherein the assessing of the fold change in afactor indicative of disease burden between two time points comprises acomparison to a threshold value, wherein the comparison indicates therisk or likely risk of the subject developing toxicity followingadministration of the cell therapy to the subject.
 50. The method of anyof claims 28-49, wherein the fold change in the factor indicative ofdisease burden correlates to a risk that the subject is or is likely todevelop toxicity following administration of the cell therapy when it isadministered.
 51. The method of any of claims 28-50, wherein: thesubject is or is likely at risk of developing toxicity if the foldchange is at or above a threshold value; or the subject is not or islikely not at risk of developing toxicity if the fold change is below athreshold value.
 52. The method of claim 51, wherein the threshold valueis a value that: i) is within 25%, within 20%, within 15%, within 10%,or within 5% above the average fold change of the factor indicative ofdisease burden and/or is within a standard deviation above the averagefold change of the factor indicative of disease burden in a plurality ofcontrol subjects; ii) is above the highest fold change of the factorindicative of disease burden, optionally within 50%, within 25%, within20%, within 15%, within 10%, or within 5% above such highest foldchange, measured in at least one subject from among a plurality ofcontrol subjects; and/or iii) is above the highest fold change asmeasured among more than 75%, 80%, 85%, 90%, or 95%, or 98% of subjectsfrom a plurality of control subjects.
 53. The method of claim 52,wherein the plurality of control subjects are a group of subjects priorto receiving a cell therapy for treating a disease or condition, saidcell therapy containing cells genetically engineered to express arecombinant receptor, wherein each of the subjects of the group went onto develop toxicity, optionally early toxicity, optionally a fever or asustained fever, within 7 days after receiving the cell therapy fortreating the same disease or condition.
 54. The method of any of claims28-53, wherein if the assessing indicates the subject is or is likely todevelop toxicity following administration of the cell therapy, thetherapeutic regimen comprises administering to the subject: i. an agentor other treatment capable of treating, preventing, delaying, reducingor attenuating the development or risk of development of a toxicity andthe cell therapy, wherein administration of the agent is to beadministered (i) prior to, (ii) within one, two, or three days of, (iii)concurrently with and/or (iv) at first fever following, the initiationof administration of the cell therapy to the subject; ii. the celltherapy at a reduced dose or at a dose that is not associated with riskof developing toxicity or severe toxicity, or is not associated with arisk of developing a toxicity or severe toxicity in a majority ofsubjects, and/or a majority of subjects having a disease or conditionthat the subject has or is suspected of having, following administrationof the cell therapy; and/or iii. the cell therapy in an in-patientsetting and/or with admission to the hospital for one or more days,optionally wherein the cell therapy is otherwise to be administered tosubjects on an outpatient basis or without admission to the hospital forone or more days; or iv. an alternative therapeutic treatment other thanthe cell therapy.
 55. The method of any of claims 28-53, wherein if theassessing indicates the subject is not or is likely not to developtoxicity following administration of the cell therapy, the therapeuticregimen comprises administering to the subject: i. the cell therapy,optionally at a non-reduced dose, optionally on an outpatient basis orwithout admission to the hospital for one or more days; ii. the celltherapy, wherein administration of the cell therapy does not compriseadministering, prior to or concurrently with administering the celltherapy and/or prior to the development of a sign or symptom of toxicityother than fever, an agent or other treatment capable of treating,preventing, delaying, or attenuating the development of the toxicity; oriii. the cell therapy in an outpatient setting and/or without admissionof the subject to the hospital overnight or for one or more consecutivedays and/or is without admission of the subject to the hospital for oneor more days.
 56. The method of any of claims 28-53, wherein if theassessing indicates the subject is or is likely to develop toxicityfollowing administration of the cell therapy, the cell therapy is notadministered to the subject.
 57. The method of any of claims 1-56,wherein the disease or condition is a cancer.
 58. The method of claim57, wherein the cancer is a myeloma, lymphoma or leukemia.
 59. Themethod of any of claims 1-58, wherein the disease or condition is a Bcell malignancy.
 60. The method of claim 59, wherein the B cellmalignancy is selected from acute lymphoblastic leukemia (ALL), chroniclymphoblastic leukemia (CLL), acute myelogenous leukemia (AML), chronicmyelogenous leukemia (CML), non-Hodgkin lymphoma (NHL), or Diffuse LargeB-Cell Lymphoma (DLBCL), or a subtype of any of the foregoing.
 61. Themethod of any of claims 27-60, wherein: greater than or greater thanabout 30%, 35%, 40%, or 50% of the subjects treated according to themethod do not exhibit any grade of cytokine release syndrome (CRS) orneurotoxicity; and/or at least at or about 45, 50, 60, 65, 70, 75, 80,85, 90, 95% or about 100% of subjects treated according to the method donot exhibit severe CRS, optionally grade 3 or higher, prolonged grade 3or higher or grade 4 or 5 CRS; and/or at least at or about 45, 50, 60,65, 70, 75, 80, 85, 90, 95% or about 100% of subjects treated accordingto the method do not exhibit severe neurotoxicity, optionally grade 3 orhigher, prolonged grade 3 or higher or grade 4 or 5 neurotoxicity;and/or at least at or about 45, 50, 60, 65, 70, 75, 80, 85, 90, 95% orabout 100% of subjects treated according to the method do not exhibitcerebral edema.
 62. The method of any of claims 27-61, wherein: prior toinitiation of administration of the dose of cells, the subject has notbeen administered an agent or other treatment capable of treating,preventing, delaying, reducing or attenuating the development or risk ofdevelopment of a toxicity; and/or the subject is not administered anagent or other treatment for the treatment or prevention or reduction orattenuation of a neurotoxicity and/or a cytokine release syndrome orrisk thereof, within a period of time following administration of thedose, which period of time is optionally at or about 1, 2, 3, 4, 5 daysor is optionally at or about 6, 7, 8, 9, 10, 11 days or is optionally 1or 2 or 3 or 4 weeks; and/or the subject is not administered an agent orother treatment for the treatment or prevention or reduction orattenuation of a neurotoxicity and/or a cytokine release syndrome orrisk thereof, following administration of the dose, prior to or unlessthe subject exhibits a sign or symptom of the toxicity and/or prior toor unless the subject exhibits a sign or symptom of the toxicity otherthan a fever, optionally wherein the fever is not a sustained fever orthe fever is or has been reduced or reduced by more than 1C aftertreatment with an antipyretic; and/or the administration and anyfollow-up is carried out on an outpatient basis and/or without admittingthe subject to a hospital and/or without an overnight stay at a hospitaland/or without requiring admission to or an overnight stay at ahospital, optionally unless or until the subject exhibits a sustainedfever or a fever that is or has not been reduced or not reduced by morethan 1C after treatment with an antipyretic.
 63. The method of any ofclaims 27-62, wherein: prior to initiation of administration of the doseof cells, the subject has not been administered an anti-IL-6 oranti-IL-6R antibody, optionally tocilizumab or siltuximab, and/or hasnot been administered a steroid, optionally dexamethasone; and/or thesubject is not administered an anti-IL-6 or anti-IL-6R antibody,optionally tocilizumab or siltuximab, and/or has not been administered asteroid, optionally dexamethasone, within a period of time followingadministration of the dose, which period of time is optionally at orabout 1, 2, 3, 4, 5 days or is optionally at or about 6, 7, 8, 9, 10, 11days or is optionally 1 or 2 or 3 or 4 weeks; and/or the subject is notadministered an anti-IL-6 or anti-IL-6R antibody, optionally tocilizumabor siltuximab, and/or has not been administered a steroid, optionallydexamethasone, following administration of the cell dose, prior to, orunless, the subject exhibits a sign or symptom of a toxicity, optionallya neurotoxicity or CRS, and/or prior to, or unless, the subject exhibitsa sign or symptom of a toxicity, optionally a neurotoxicity or CRS,other than a fever, optionally wherein the fever is not a sustainedfever or the fever is or has been reduced or reduced by more than 1Cafter treatment with an antipyretic; and/or the administration and anyfollow-up is carried out on an outpatient basis and/or without admittingthe subject to a hospital and/or without an overnight stay at a hospitaland/or without requiring admission to or an overnight stay at ahospital, optionally unless or until the subject exhibits a sustainedfever or a fever that is or has not been reduced or not reduced by morethan 1° C. after treatment with an antipyretic.
 64. The method of any ofclaims 27-63, wherein: the administration is carried out on anoutpatient basis and/or without requiring admission to or an overnightstay at a hospital; and if the subject exhibits a sustained fever or afever that is or has not been reduced or not reduced by more than 1° C.after treatment with an antipyretic, the subject is admitted to thehospital or to an overnight stay at a hospital and/or is administered anagent or other treatment for the treatment or prevention or reduction orattenuation of a neurotoxicity and/or a cytokine release syndrome orrisk thereof.
 65. The method of any of claims 1-64, wherein the celltherapy is or comprises tumor infiltrating lymphocytic (TIL) therapy.66. The method of any of claims 1-65, wherein the cell therapy comprisescells engineered to express a recombinant receptor that specificallybinds to an antigen associated with a disease or condition and/orexpressed in cells associated with the disease or condition.
 67. Themethod of any of claims 1-66, wherein the cell therapy is an adoptivecell therapy.
 68. The method of claim 66 or claim 67, wherein the cellscomprise immune cells.
 69. The method of claim 68, wherein the immunecells are or comprise T cells or NK cells.
 70. The method of claim 69,wherein the immune cells are or comprise T cells and the T cellscomprise CD4+ and/or CD8+ T cells.
 71. The method of any of claims 1-70,wherein the cell therapy is a T cell therapy comprising geneticallyengineered cells expressing a recombinant receptor.
 72. The method ofclaim 71, wherein the recombinant receptor is a T cell receptor or afunctional non-T cell receptor.
 73. The method of claim 71 or claim 72,wherein the recombinant receptor specifically binds to an antigenassociated with a disease or condition and/or expressed in cellsassociated with the disease or condition.
 74. The method of claim 73,wherein the antigen is selected from among Trophoblast glycoprotein(TPBG also known as 5T4), 8H9, αvβ6 integrin (avb6 integrin), B7-H3,B7-H6, B cell maturation antigen (BCMA), carbonic anhydrase 9 (CA9, alsoknown as CAIX or G250), a cancer-testes antigen, carbonic anhydrase 9(CAIX), C-C Motif Chemokine Ligand 1 (CCL-1), CD19, CD20, CD22,hepatitis B surface antigen, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6,CD44v7/8, CD123, CD133, CD138, CD171, carcinoembryonic antigen (CEA), acyclin, cyclin A2, c-Met, dual antigen, chondroitin sulfate proteoglycan4 (CSPG4), epidermal growth factor protein (EGFR), epithelialglycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrinB2,ephrin receptor A2 (EPHa2), ephrinB2, type III epidermal growth factorreceptor mutation (EGFR vIII), estrogen receptor, folate receptor alpha,folate binding protein (FBP), Fc receptor like 5 (FCRL5; also known asFc receptor homolog 5 or FCRH5), fetal acetylcholine receptor (FetalAchR), G250/CAIX, ganglioside GD2, ganglioside GD3, glycoprotein 100(gp100), glypican-3 (GPC3), G Protein Coupled Receptor 5D (GPRC5D),Her2/neu (receptor tyrosine kinase erbB2), Her3 (erb-B3), Her4 (erb-B4),erbB dimers, Human high molecular weight-melanoma-associated antigen(HMW-MAA), Human leukocyte antigen A1 (HLA-A1), Human leukocyte antigenA2 (HLA-A2), IL-22 receptor alpha (IL-22R-alpha), IL-13 receptor alpha 2(IL-13Ra2), kinase insert domain receptor (kdr), kappa light chain,Leucine Rich Repeat Containing 8 Family Member A (LRRC8A), Lewis Y,L1-cell adhesion molecule (L1-CAM), CE7 epitope of L1-CAM,Melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MAGE-A10, melanA (MART-1), mesothelin (MSLN), murine cytomegalovirus (CMV), mucin 1(MUC1), MUC16, neural cell adhesion molecule (NCAM), natural killergroup 2 member D (NKG2D), NKG2D ligands, cancer/testis antigen 1B (CTAG,also known as NY-ESO-1 and LAGE-2), O-acetylated GD2 (OGD2), oncofetalantigen, Preferentially expressed antigen of melanoma (PRAME), aprostate specific antigen, prostate stem cell antigen (PSCA),progesterone receptor, survivin, prostate specific membrane antigen(PSMA), Receptor Tyrosine Kinase Like Orphan Receptor 1 (ROR1),tumor-associated glycoprotein 72 (TAG72), Tyrosinase related protein 1(TRP1, also known as TYRP1 or gp75), Tyrosinase related protein 2 (TRP2,also known as dopachrome tautomerase, dopachrome delta-isomerase orDCT), vascular endothelial growth factor receptor (VEGF receptors;VEGFR), vascular endothelial growth factor receptor 2 (VEGF-R2), WilmsTumor 1 (WT-1), a pathogen-specific antigen.
 75. The method of any ofclaims 71-74, wherein the recombinant receptor is a chimeric antigenreceptor (CAR).
 76. The method of claim 75, wherein the CAR comprises anextracellular antigen-recognition domain that specifically binds to theantigen and an intracellular signaling domain comprising an ITAM,wherein optionally, the intracellular signaling domain comprises anintracellular domain of a CD3-zeta (CD3ζ) chain; and/or wherein the CARfurther comprises a costimulatory signaling region, which optionallycomprises a signaling domain of CD28 or 4-1BB.
 77. The method of any ofclaims 71-76, wherein the genetically engineered cells comprise T cellsor NK cells.
 78. The method of any of claims 71-77, wherein thegenetically engineered cells comprise T cells, and the T cells compriseCD4+ and/or CD8+ T cells.
 79. The method of claim 78, wherein the Tcells are primary T cells obtained from a subject.
 80. The method of anyof claims 1-79, wherein the cells of the cell therapy are autologous tothe subject or the cells are allogeneic to the subject.
 81. The methodof any of claims 21-80, wherein the threshold value is a threshold valuefor fold change of a volumetric measure of disease burden that is SPD,wherein the fold change is at least about or is about or is 5 fold, 6fold, 7 fold, 8 fold, or 9 fold.
 82. The method of any of claims 1-81,wherein the cell therapy comprises the administration of from or fromabout 1×10⁵ to 1×10⁸ total recombinant receptor-expressing cells, totalT cells, or total peripheral blood mononuclear cells (PBMCs), from orfrom about 5×10⁵ to 1×10⁷ total recombinant receptor-expressing cells,total T cells, or total peripheral blood mononuclear cells (PBMCs) orfrom or from about 1×10⁶ to 1×10⁷ total recombinant receptor-expressingcells, total T cells, or total peripheral blood mononuclear cells(PBMCs), each inclusive.
 83. The method of any of claims 1-82, whereinthe cell therapy comprises the administration of no more than 1×10⁸total recombinant receptor-expressing cells, total T cells, or totalperipheral blood mononuclear cells (PBMCs), no more than 1×10⁷ totalrecombinant receptor-expressing cells, total T cells, or totalperipheral blood mononuclear cells (PBMCs), no more than 0.5×10⁷ totalrecombinant receptor-expressing cells, total T cells, or totalperipheral blood mononuclear cells (PBMCs), no more than 1×10⁶ totalrecombinant receptor-expressing cells, total T cells, or totalperipheral blood mononuclear cells (PBMCs), no more than 0.5×10⁶ totalrecombinant receptor-expressing cells, total T cells, or totalperipheral blood mononuclear cells (PBMCs).
 84. A kit, comprising (a) acell therapy, said cell therapy comprising cells genetically engineeredto express a recombinant receptor; and (b) instructions foradministering the cell therapy to a subject having or suspected ofhaving a tumor, wherein the instructions specify: (1) assessing, at twodifferent time points, one or more factors indicative of disease burdenin a subject that is a candidate for treatment with the cell therapy,wherein, at each of the two time points, the subject has not yet beenadministered the cell therapy and the factors indicative of diseaseburden comprises a volumetric measure of the tumor or a level or amountof an inflammatory marker in a sample from the subject, wherein the foldchange in the factor assessed at the two different time points indicatesthe degree of risk or likely risk of the subject developing toxicityfollowing administration of the cell therapy to the subject; and (2) (i)if the fold change is at or below a threshold value, administering thecell therapy to the subject, or (ii) administering the cell therapy tothe subject, or (iii) administering the cell therapy to the subject atan amount, dose, setting, time or frequency that is based on the foldchange.
 85. A kit, comprising (a) a cell therapy, said cell therapycomprising cells genetically engineered to express a recombinantreceptor; and (b) one or more reagents for assaying one or more factorsindicative of disease burden, wherein the one or more factors indicativeof disease burden comprise an inflammatory marker selected fromC-reactive protein (CRP), erythrocyte sedimentation rate (ESR), albumin,ferritin, β2 microglobulin (β2-M), or lactate dehydrogenase (LDH). 86.The kit of claim 85, wherein the kit further comprises instructions forassessing the presence, level or amount of at least one of theinflammatory marker in two or more samples obtained at two or more timepoints from a subject that is a candidate for treatment with the celltherapy and determining a fold-change in the level or amount of themarker assessed at two of the two or more time points, wherein the foldchange indicates the degree of risk or likely risk of the subjectdeveloping toxicity following administration of the cell therapy to thesubject.
 87. The kit of claim 86, wherein the kit further comprisesinstructions for (i) administering a therapeutic regimen comprising thecell therapy to the subject if the fold change is at or below athreshold value, (ii) administering the cell therapy to the subject, or(iii) administering the therapeutic regimen to the subject at an amount,dose, setting, time or frequency based on the fold change of the atleast one inflammatory marker.
 88. A kit, comprising (a) a cell therapy,said cell therapy comprising cells genetically engineered to express arecombinant receptor; and (b) instructions for administering atherapeutic regimen comprising the cell therapy based on the results ofassessing the subject for a fold change in a factor indicative ofdisease burden between two time points prior to receiving a celltherapy, wherein the instructions specify (i) administering the celltherapy to the subject if the fold change is at or below a thresholdvalue, (ii) administering the cell therapy to the subject, or (iii)administering the therapeutic regimen to the subject at an amount, dose,setting, time or frequency based on the fold change, and wherein thesubject is a candidate for treatment with the cell therapy.
 89. The kitof claim 88, wherein the factor indicative of disease burden is avolumetric measure of a tumor or is an inflammatory marker in a samplefrom a subject.
 90. The kit of any of claim 84, 88, or 89, wherein thefactor indicative of disease burden is a volumetric measure and thevolumetric measure is a sum of the products of diameters (SPD), longesttumor diameters (LD), sum of longest tumor diameters (SLD), necrosis,tumor volume, necrosis volume, necrosis-tumor ratio (NTR), peritumoraledema (PTE), or edema-tumor ratio (ETR).
 91. The kit of any of claim 84,or 89-90, wherein the volumetric measure is assessed using computedtomography (CT), positron emission tomography (PET), and/or magneticresonance imaging (MRI) of the subject.
 92. The kit of any of claims84-89, wherein the kit comprises reagents for detecting C-reactiveprotein (CRP), erythrocyte sedimentation rate (ESR), albumin, ferritin,β2 microglobulin (β2-M), or lactate dehydrogenase (LDH).
 93. The kit ofany of claim 84-89 or 92, wherein the inflammatory marker is assessedusing a colorimetric assay or an immunoassay.
 94. The kit of any ofclaim 84-89 or 92-93, wherein the inflammatory marker is assessed usingan immunoassay and the immunoassay is selected from enzyme-linkedimmunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay(RIA), surface plasmon resonance (SPR), Western Blot, Lateral flowassay, immunohistochemistry, protein array or immuno-PCR (iPCR).
 95. Thekit of any of claim 84-89 or 92-94, wherein the sample is or comprises ablood sample, plasma sample, or serum sample.
 96. The kit of any ofclaims 84-95, wherein the subject is a human.
 97. The kit of any ofclaim 84 or 86-96, wherein the two time points comprises a first timepoint and a second time point, and wherein the fold change is a ratio ofthe factor indicative of disease burden at the first time point and thefactor indicative of disease burden at the second time point.
 98. Thekit of any of claim 84 or 86-97, wherein, if the cell therapy is to beadministered to the subject, the two time points are both no more thanone month or two months prior to the subject receiving the cell therapy.99. The kit of any of claims 84-98 wherein the two time points are notless than one week, two weeks, three weeks, four weeks, or five weeksapart.
 100. The kit of any of claims 84-99, wherein the two time pointsare not less than three weeks apart.
 101. The kit of any of claims84-100 wherein the two time points are not more than four weeks apart,five weeks, or six weeks apart.
 102. The kit of any of claims 84-101,wherein, if the cell therapy is to be administered to the subject, thesecond time point is more than 1, 2, 3, 4, 5, 6, or 7 days beforeadministration of the cell therapy.
 103. The kit of any of claim 84 or87-102, wherein: the instructions specify that the fold change in thefactor indicative of disease burden indicates the subject is or islikely at risk of developing toxicity if the fold change is at or abovethe threshold value; or the instructions specify that the fold change inthe factor indicative of disease burden indicates the subject is not oris likely not at risk of developing toxicity if the fold change is belowthe threshold value.
 104. The kit of any of claim 84 or 87-103, whereinthe threshold value is a value that: i) is within 25%, within 20%,within 15%, within 10%, or within 5% above the average fold change ofthe factor indicative of disease burden and/or is within a standarddeviation above the average fold change of the factor indicative ofdisease burden in a plurality of control subjects; ii) is above thehighest fold change of the factor indicative of disease burden,optionally within 50%, within 25%, within 20%, within 15%, within 10%,or within 5% above such highest fold change, as assessed in at least onesubject from among a plurality of control subjects; and/or iii) is abovethe highest fold change as assessed among more than 75%, 80%, 85%, 90%,or 95%, or 98% of subjects from a plurality of control subjects. 105.The kit of claim 104, wherein the plurality of control subjects is agroup of subjects having been administered a cell therapy for treating adisease or condition and having had the fold change of the factorindicative of disease burden assessed, wherein each of the subjects ofthe group went on to develop toxicity, optionally early toxicity,optionally a fever or a sustained fever, within 7 days after receivingthe cell therapy for treating the same disease or condition.
 106. Thekit of any of claim 84 or 86-105, wherein the instructions furtherspecify, if the fold change indicates that the subject is or is likelyat risk of developing toxicity, selecting the subject for administrationof a therapeutic regimen, the therapeutic regimen comprisingadministering to the subject: i. an agent or other treatment capable oftreating, preventing, delaying, reducing or attenuating the developmentor risk of development of a toxicity and the cell therapy, whereinadministration of the agent is to be administered (i) prior to, (ii)within one, two, or three days of, (iii) concurrently with and/or (iv)at first fever following, the initiation of administration of the celltherapy to the subject; ii. the cell therapy at a reduced dose or at adose that is not associated with risk of developing toxicity or severetoxicity, or is not associated with a risk of developing a toxicity orsevere toxicity in a majority of subjects, and/or a majority of subjectshaving a disease or condition that the subject has or is suspected ofhaving, following administration of the cell therapy; and/or iii. thecell therapy in an in-patient setting and/or with admission to thehospital for one or more days, optionally wherein the cell therapy isotherwise to be administered to subjects on an outpatient basis orwithout admission to the hospital for one or more days; or iv. analternative therapeutic treatment other than the cell therapy.
 107. Thekit of any of claim 84 or 86-105, wherein the instructions furtherspecify, if the fold change indicates that the subject is not or islikely not at risk of developing toxicity, selecting the subject foradministration of a therapeutic regimen, the therapeutic regimencomprising administering to the subject: i. the cell therapy, optionallyat a non-reduced dose, optionally on an outpatient basis or withoutadmission to the hospital for one or more days; ii. the cell therapy,wherein administration of the cell therapy does not compriseadministering, prior to or concurrently with administering the celltherapy and/or prior to the development of a sign or symptom of toxicityother than fever, an agent or other treatment capable of treating,preventing, delaying, or attenuating the development of the toxicity; oriii. the cell therapy in an outpatient setting and/or without admissionof the subject to the hospital overnight or for one or more consecutivedays and/or is without admission of the subject to the hospital for oneor more days.
 108. The kit of any of claim 84 or 86-105, wherein theinstructions further specify that, if the fold change indicates that thesubject is or is likely at risk of developing toxicity, the therapeuticregimen is not to be administered to the subject.
 109. The kit of claim107, wherein: the administration is carried out on an outpatient basisand/or without requiring admission to or an overnight stay at ahospital; and if the subject exhibits a sustained fever or a fever thatis or has not been reduced or not reduced by more than 1° C. aftertreatment with an antipyretic, the subject is admitted to the hospitalor to an overnight stay at a hospital and/or is administered an agent orother treatment for the treatment or prevention or reduction orattenuation of a neurotoxicity and/or a cytokine release syndrome orrisk thereof.
 110. The kit of any of claim 84 or 86-109, wherein thetoxicity is neurotoxicity and/or cytokine release syndrome (CRS). 111.The kit of any of claim 84 or 86-110, wherein the toxicity is earlytoxicity that develops within 7 days of administration of the celltherapy.
 112. The kit of any of claim 84 or 86-111, wherein the toxicitydevelops within 3, 4, 5, 6, or 7 days of administration of the celltherapy.
 113. The kit of any of claim 84 or 86-112, wherein the toxicityis a first sign of a fever or is a sustained fever followingadministration of the cell therapy.
 114. The kit of any of claims84-113, wherein the cell therapy is or comprises tumor infiltratinglymphocytic (TIL) therapy.
 115. The kit of any of claims 84-114, whereinthe cell therapy comprises cells engineered to express a recombinantreceptor that specifically binds to an antigen associated with a diseaseor condition and/or expressed in cells associated with the disease orcondition.
 116. The kit of any of claims 84-115, wherein the celltherapy is an adoptive cell therapy.
 117. The kit of claim 115 or claim116, wherein the cells comprise immune cells.
 118. The kit of claim 117,wherein the immune cells are or comprise T cells or NK cells.
 119. Thekit of claim 118, wherein the immune cells are or comprise T cells andthe T cells comprise CD4+ and/or CD8+ T cells.
 120. The kit of any ofclaims 84-119, wherein the cell therapy is a T cell therapy comprisinggenetically engineered cells expressing a recombinant receptor.
 121. Thekit of any of claims 105-120, wherein the disease or condition is acancer.
 122. The kit of claim 121, wherein the cancer is a myeloma,leukemia or lymphoma.
 123. The kit of any of claims 105-120, wherein thedisease or condition is a B cell malignancy.
 124. The kit of claim 123,wherein the B cell malignancy is selected from acute lymphoblasticleukemia (ALL), chronic lymphoblastic leukemia (CLL), acute myelogenousleukemia (AML), chronic myelogenous leukemia (CML), non-Hodgkin lymphoma(NHL), or Diffuse Large B-Cell Lymphoma (DLBCL), or a subtype of any ofthe foregoing.
 125. The kit of any of claims 115-124, wherein theantigen is selected from among Trophoblast glycoprotein (TPBG also knownas 5T4), 8H9, αvβ6 integrin (avb6 integrin), B7-H3, B7-H6, B cellmaturation antigen (BCMA), carbonic anhydrase 9 (CA9, also known as CAIXor G250), a cancer-testes antigen, carbonic anhydrase 9 (CAIX), C-CMotif Chemokine Ligand 1 (CCL-1), CD19, CD20, CD22, hepatitis B surfaceantigen, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD123,CD133, CD138, CD171, carcinoembryonic antigen (CEA), a cyclin, cyclinA2, c-Met, dual antigen, chondroitin sulfate proteoglycan 4 (CSPG4),epidermal growth factor protein (EGFR), epithelial glycoprotein 2(EPG-2), epithelial glycoprotein 40 (EPG-40), ephrinB2, ephrin receptorA2 (EPHa2), ephrinB2, type III epidermal growth factor receptor mutation(EGFR vIII), estrogen receptor, folate receptor alpha, folate bindingprotein (FBP), Fc receptor like 5 (FCRL5; also known as Fc receptorhomolog 5 or FCRH5), fetal acetylcholine receptor (Fetal AchR),G250/CAIX, ganglioside GD2, ganglioside GD3, glycoprotein 100 (gp100),glypican-3 (GPC3), G Protein Coupled Receptor 5D (GPRC5D), Her2/neu(receptor tyrosine kinase erbB2), Her3 (erb-B3), Her4 (erb-B4), erbBdimers, Human high molecular weight-melanoma-associated antigen(HMW-MAA), Human leukocyte antigen A1 (HLA-A1), Human leukocyte antigenA2 (HLA-A2), IL-22 receptor alpha (IL-22R-alpha), IL-13 receptor alpha 2(IL-13Ra2), kinase insert domain receptor (kdr), kappa light chain,Leucine Rich Repeat Containing 8 Family Member A (LRRC8A), Lewis Y,L1-cell adhesion molecule (L1-CAM), CE7 epitope of L-CAM,Melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MAGE-A10, melanA (MART-1), mesothelin (MSLN), murine cytomegalovirus (CMV), mucin 1(MUC1), MUC16, neural cell adhesion molecule (NCAM), natural killergroup 2 member D (NKG2D), NKG2D ligands, cancer/testis antigen 1B (CTAG,also known as NY-ESO-1 and LAGE-2), O-acetylated GD2 (OGD2), oncofetalantigen, Preferentially expressed antigen of melanoma (PRAME), aprostate specific antigen, prostate stem cell antigen (PSCA),progesterone receptor, survivin, prostate specific membrane antigen(PSMA), Receptor Tyrosine Kinase Like Orphan Receptor 1 (ROR1),tumor-associated glycoprotein 72 (TAG72), Tyrosinase related protein 1(TRP1, also known as TYRP1 or gp75), Tyrosinase related protein 2 (TRP2,also known as dopachrome tautomerase, dopachrome delta-isomerase orDCT), vascular endothelial growth factor receptor (VEGF receptors;VEGFR), vascular endothelial growth factor receptor 2 (VEGF-R2), WilmsTumor 1 (WT-1), a pathogen-specific antigen.
 126. The kit of any ofclaims 84-125, wherein the recombinant receptor is a T cell receptor ora functional non-T cell receptor.
 127. The kit of any of claims 84-126,wherein the recombinant receptor is a chimeric antigen receptor (CAR).128. The kit of claim 127, wherein the CAR comprises an extracellularantigen-recognition domain that specifically binds to the antigen and anintracellular signaling domain comprising an ITAM, wherein optionally,the intracellular signaling domain comprises an intracellular domain ofa CD3-zeta (CD3ζ) chain; and/or wherein the CAR further comprises acostimulatory signaling region, which optionally comprises a signalingdomain of CD28 or 4-1BB.
 129. The kit of any of claims 84-128, furthercomprising an agent or other treatment capable of treating, preventing,delaying, reducing or attenuating the development or risk of developmentof a toxicity.
 130. The kit of claim 129, wherein the agent or othertreatment is or comprises one or more of a steroid; an antagonist orinhibitor of a cytokine receptor or cytokine selected from among IL-10,IL-10R, IL-6, IL-6 receptor, IFN7, IFNGR, IL-2, IL-2R/CD25, MCP-1, CCR2,CCR4, MIP1β, CCR5, TNFalpha, TNFR1, IL-1, and IL-1Ralpha/IL-1beta; or anagent capable of preventing, blocking or reducing microglial cellactivity or function.
 131. The kit of claim 130, wherein the antagonistor inhibitor is or comprises an agent selected from among an antibody orantigen-binding fragment, a small molecule, a protein or peptide and anucleic acid.
 132. The kit of any of claims 129-131, wherein the agentor other treatment is an anti-IL-6 antibody or an anti-IL6 receptorantibody.
 133. The kit of any of claims 129-132, wherein the agent orother treatment is or comprises an agent selected from amongtocilizumab, siltuximab, clazakizumab, sarilumab, olokizumab (CDP6038),elsilimomab, ALD518/BMS-945429, sirukumab (CNTO 136), CPSI-2634,ARGX-109, FE301 and FM101.
 134. The kit of any of claims 129-133,wherein the agent or other treatment is or comprises tocilizumab. 135.The kit of any of claims 129-134, wherein the agent or other treatmentis or comprises siltuximab.
 136. The kit of any of claims 130-135,wherein the steroid is or comprises dexamethasone.
 137. The kit of anyof claims 84-136, wherein the genetically engineered cells comprise Tcells, and the T cells comprise CD4+ and/or CD8+ T cells.
 138. The kitof claim 137, wherein the T cells are primary T cells obtained from asubject.
 139. The kit of any of claims 84-138, wherein the cells of thecell therapy are autologous to the subject.
 140. The kit of any ofclaims 84-138, wherein the cells of the cell therapy are allogeneic tothe subject.
 141. The kit of any of claims 103-140, wherein thethreshold value is a threshold value for fold change of a volumetricmeasure of disease burden that is SPD, wherein the fold change is atleast about or is about or is 5 fold, 6 fold, 7 fold, 8 fold, or 9 fold.